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The type III calcium signaling mechanism of CD38
Abstract
CD38 is a membrane protein consisting of a catalytic carboxyl domain (C), a single stretch of trans-membrane segment and a short amino (N) tail of 21 residues. It has long been thought to have a type II membrane orientation with the catalytic C-domain facing outside. It has never been
fully understood how an ecto-enzyme can gain access to its intracellular substrates, NAD and NADP, and produce the signaling messengers, cADPR and NAADP that target intracellular Ca2+ stores. A vesicular mechanism has been proposed, positing that CD38 can be endocytosed, together
with transport proteins, into endo-lysosomes. These proteins, such as connexin 43 and nucleoside transporters, can then mediate the influx of substrates for CD38 and efflux of the messenger products from the endo-lysosomes. Recently, we have shown that CD38, in fact, exists naturally in two
opposite membrane orientations in human peripheral blood mononuclear cells, promyelocytic leukemic HL60 cells and the monocytic U937 cells, not only in the expected type II orientation but also in the opposite type III orientation, with the catalytic C-domain facing the cytosol (Science Signaling
5, ra67 (2012)). The type III CD38 is fully active in producing intracellular cADPR. This presentation will summarize and update the current status of the topological aspects of CD38-signaling mechanism.
... 36−39 Like the most common type II CD38, type III CD38 is functionally active in producing the calcium-mobilizing messengers ( Figure 1B). 36,37 The membrane-bound CD38 is characterized by robust NAD + -glycohydrolase (NADase) activity and plays a major role in modulating NAD + levels in tissues and organs. 11,13,40−43 Genetic deletion or pharmacological inhib-ition of CD38 provides protection from aging-related metabolic disorders, myocardial infarction, nonalcoholic fatty liver disease, neuroinflammation, and fibrosis in preclinical models by augmenting NAD + levels in vivo. ...
Nicotinamide adenine dinucleotide (NAD⁺) is required for a myriad of metabolic, signaling, and post-translational events in cells. Its levels in tissues and organs are closely associated with health conditions. The homeostasis of NAD⁺ is regulated by biosynthetic pathways and consuming enzymes. As a membrane-bound protein with robust NAD⁺ hydrolase activity, cluster of differentiation 38 (CD38) is a major degrader of NAD⁺. Deficiency or inhibition of CD38 enhances NAD⁺ levels in vivo, resulting in various therapeutic benefits. As a metabolic precursor of NAD⁺, nicotinamide mononucleotide can be rapidly hydrolyzed by CD38, whereas nicotinamide riboside (NR) lacks CD38 substrate activity. Given their structural similarities, we explored the inhibition potential of NR. To our surprise, NR exhibits marked inhibitory activity against CD38 by forming a stable ribosyl–ester bond with the glutamate residue 226 at the active site. Inspired by this discovery, we designed and synthesized a clickable NR featuring an azido substitution at the 5′-OH position. This cell-permeable NR analogue enables covalent labeling and imaging of both extracellular and intracellular CD38 in live cells. Our work discovers an unrecognized molecular function of NR and generates a covalent probe for health-related CD38. These findings offer new insights into the role of NR in modulating NAD⁺ metabolism and CD38-mediated signaling as well as an innovative tool for in-depth studies of CD38 in physiology and pathophysiology.
... We further showed that the type III CD38 is functionally active in producing cellular cADPR, the levels of which directly correlate with the CIB1 levels. The results substantiate the type III signaling mechanism of CD38 (1,18). ...
Significance
This study addresses a topological paradox in Ca ²⁺ signaling mediated by cyclic ADP-ribose (cADPR). The messenger is synthesized by CD38, thought to be a type ll ectoenzyme. Instead, we show that it exists naturally in two opposite membrane orientations and that the type III, with its catalytic domain facing the cytosol, is active in producing cellular cADPR. Its topology was resolved by a technique simultaneously targeting dual epitopes for protein identification, which identifies the intracellular type III CD38 unambiguously. It is also shown that its cADPR-synthesizing activity is regulated by cytosolic interactions with Ca ²⁺ and integrin-binding protein 1 (CIB1). The results indicate that membrane proteins are not necessarily expressed with only one unique membrane orientation set by sequence motifs as generally believed.
CD38 is a single-pass transmembrane enzyme catalyzing the synthesis of two nucleotide second messengers, cyclic ADP-ribose (cADPR) from NAD and nicotinic acid adenine dinucleotide phosphate (NAADP) from NADP. The former mediates the mobilization of the endoplasmic Ca²⁺-stores in response to a wide range of stimuli, while NAADP targets the endo-lysosomal stores. CD38 not only possesses multiple enzymatic activities, it also exists in two opposite membrane orientations. Type III CD38 has the catalytic domain facing the cytosol and is responsible for producing cellular cADPR. The type II CD38 has an opposite orientation and is serving as a surface receptor mediating extracellular functions such as cell adhesion and lymphocyte activation. Its ecto-NADase activity also contributes to the recycling of external NAD released by apoptosis. Endocytosis can deliver surface type II CD38 to endo-lysosomes, which acidic environment favors the production of NAADP. This article reviews the rationale and evidence that have led to CD38 as a paradigm for membrane topology defining distinct functions of proteins. Also described is the recent discovery of a hitherto unknown cADPR-synthesizing enzyme, SARM1, ushering in a new frontier in cADPR-mediated Ca²⁺-signaling.
Oxytocin (OT) is a critical molecule for social recognition that mediates social memory and emotional behaviors. OT is known to be released during stress and acts as an anxiolytic factor. Recent studies revealed higher levels of OT release in hypothalamus culture isolated from subordinate mice in group-housed males than in that from dominant mice after cage-switch stress. OT concentrations in micro-perfusates at the paraventricular nucleus upon perfusion stimulation with cADPR were enhanced in subordinate mice compared with dominant mice. OT concentration in cerebrospinal fluid was higher in endotoxin-shock mice with fever than in controls without an increase in body temperature. In mice exposed to the new environmental stress, the CSF OT level transiently increased at 5 min from start of exposure, while the rectal temperature also increased. OT release under various conditions was sensitive to antagonists, gene knockout, or mRNA levels of CD38 or TRPM2 in the hypothalamus. These findings indicated that cADPR and hyperthermia co-regulate hypothalamic OT secretion during social stress by elevation of intracellular free Ca²⁺ concentrations involved in CD38-dependent Ca²⁺ mobilization and TRPM2-dependent Ca²⁺ influx. Interaction between CD38 and TRPM2 seems to involve a different mechanism for stress-induced release of OT that may result in anxiolytic effects, resulting in transient rescission of autistic phenotypes.
Oxytocin (OT) is a critical molecule for social recognition that mediates social and emotional behaviors. OT is released during stress and acts as an anxiolytic factor. To know the precise molecular mechanisms underlying OT release into the brain during stress is important. It has been reported that intracellular concentrations of free calcium in the hypothalamic neurons are elevated by simultaneous stimulation of cyclic ADP-ribose (cADPR) and heat. We have reported in vitro and in vivo data that supports the idea that release of OT in the brain of male mice is regulated by cADPR and fever in relation to stress conditions. 1) Significantly higher levels of OT release were observed in hypothalamus cultures isolated from subordinate mice in group-housed males compared to dominant males after cage-switch stress; 2) OT concentrations in micro-perfusates at the paraventricular nucleus upon perfusion stimulation with cADPR were enhanced in subordinate mice compared to dominant mice; 3) The OT concentration in the cerebrospinal fluid (CSF) was higher in endotoxin-shock mice with fever compared to controls with no body temperature increase; and 4) In mice exposed to new environmental stress, the CSF OT level transiently increased 5 min after exposure, while the rectal temperature increased from 36.6 °C to 37.8 °C from 5 to 15 min after exposure. In this review, we examine whether or not cADPR and hyperthermia co-regulate hypothalamic OT secretion during social stress through the elevation of intracellular free Ca²⁺ concentrations involved in CD38-dependent Ca²⁺ mobilization and TRPM2-dependent Ca²⁺ influx. Finally, we propose that the interaction between CD38 and TRPM2 seems to be a new mechanism for stress-induced release of OT, which may result in anxiolytic effects for temporal recovery from social impairments in children with autism spectrum disorder during hyperthermia.
Using primary cultured mouse microglia, expression of CD38 was significantly up-regulated in lipopolysaccharide (LPS, 100 ng/mL)-activated microglia, but not in adenosine triphosphate (ATP, 100 μM)-treated microglia (24 h pretreatment). Since TRPM2 (transient receptor potential
cation channel, subfamily M, member 2) is highly expressed in microglia and is activated by cADPR and ADPR, the effect of knock-down of TRPM2 using siRNA on the expression of CD38 was examined in activated microglia. Unexpectedly, knock-down of TRPM2 significantly up-regulated the expression
of CD38. LPS-induced production of nitric oxide was not affected by TRPM2 siRNA, while release of TNF-α and IL-1β were attenuated by TRPM2 siRNA. These results suggest that CD38 may substitute for the expression of TRPM2 when TRPM2 is absent or decreased and partially
compensate the [Ca2+] i mobilization. Taking account of the role of CD38, especially in activated microglia, dysfunction of CD38 could disturb Ca2+ signaling in microglia as well and may lead to breakdown of the brain homeostasis.
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