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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 Ca 2+ -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 Ca 2+ -signaling., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

Background: CD38 is a surface membrane antigen highly expressed in malignant blood cells, such as multiple myeloma (MM). A soluble form of CD38 (sCD38) is also present in the plasma, deriving likely from the shedding from the cells. The plasma levels of sCD38 should thus correlate closely with the proliferation of the MM cells, allowing the development of a simple diagnostic blood test for monitoring the progress of the disease. However, the plasma sCD38 levels are extremely low, requiring the design of a highly sensitive and specific assay., Results: In this study, we developed an ultra-sensitive assay, based on two nanobodies (Nbs) targeting two distinct epitopes of sCD38. One Nb acts as a capturer, and the other is fused with the firefly luciferase serving as a reporter to ensure sensitivity. We showed that this Dual epitopes protein IDentification (DepID) assay has sensitivity reaching 10 pg/mL, which is 10 times higher than that of a commercial ELISA kit. By this method, we were able to precisely quantify the levels of sCD38 in the plasma of MM patients, which were significantly higher than those from healthy donors. We further showed that the increase plasma levels of sCD38 correlated with the progress of MM., Conclusion: We have developed a Nb-based luminescence sandwich assay, named as DepID, for quantification of the soluble CD38 in MM patients' plasma and showed the potency of this method as a tool for general diagnosis of MM or companion diagnosis of the CD38-targeted therapies., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Protein ADP-ribosylation, including mono- and poly-ADP-ribosylation, is increasingly recognized to play important roles in various biological pathways. Molecular understanding of the functions of ADP-ribosylation requires the identification of the sites of modification. Although tandem mass spectrometry (MS/MS) is widely recognized as an effective means for determining protein modifications, identification of ADP-ribosylation sites has been challenging due to the labile and hydrophilic nature of the modification. Here we applied precursor ion scanning-triggered MS/MS analysis on a hybrid quadrupole linear ion trap mass spectrometer for selectively detecting ADP-ribosylated peptides and determining the auto-ADP-ribosylation sites of CD38 (cluster of differentiation 38) E226D and E226Q mutants. CD38 is an enzyme that catalyzes the hydrolysis of nicotinamide adenine dinucleotide (NAD) to ADP-ribose. Here we show that NAD can covalently label CD38 E226D and E226Q mutants but not wild-type CD38. In this study, we have successfully identified the D226/Q226 and K129 residues of the two CD38 mutants being the ADP-ribosylation sites using precursor ion scanning hybrid quadrupole linear ion trap mass spectrometry. The results offer insights about the CD38 enzymatic reaction mechanism. The precursor ion scanning method should be useful for identifying the modification sites of other ADP-ribosyltransferases such as poly(ADP-ribose) polymerases., (Copyright © 2012 Elsevier Inc. All rights reserved.)

Cyclic ADP-ribose (cADPR) is a calcium messenger that can mobilize intracellular Ca²⁺ stores and activate Ca²⁺ influx to regulate a wide range of physiological processes. Aplysia cyclase is the first member of the ADP-ribosyl cyclases identified to catalyze the cyclization of NAD⁺ into cADPR. The catalysis involves a two-step reaction, the elimination of the nicotinamide ring and the cyclization of the intermediate resulting in the covalent attachment of the purine ring to the terminal ribose. Aplysia cyclase exhibits a high degree of leniency towards the purine base of its substrate, and the cyclization reaction takes place at either the N1- or the N7-position of the purine ring. To decipher the mechanism of cyclization in Aplysia cyclase, we used a crystallization setup with multiple Aplysia cyclase molecules present in the asymmetric unit. With the use of natural substrates and analogs, not only were we able to capture multiple snapshots during enzyme catalysis resulting in either N1 or N7 linkage of the purine ring to the terminal ribose, we were also able to observe, for the first time, the cyclized products of both N1 and N7 cyclization bound in the active site of Aplysia cyclase., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

Cellular metabolism is required for cell proliferation. However, the way in which metabolic signals are conveyed to cell cycle decisions is unclear. Cyclic ADP-ribose (cADPR), the NAD(+) metabolite, mobilizes calcium from calcium stores in many cells. We found that dinoflagellate cells with higher metabolic rate underwent multiple fission (MF), a division mode in which cells can exceed twice their sizes at G1. A temperature shift-down experiment suggested that MF involves a commitment point at late G1. In fast-growing cells, cADPR level peaked in G(1) and increased with increasing concentrations of glucose in the medium. Addition of glycolytic poison iodoacetate inhibited cell growth, reduced cADPR levels as well as the commitment of cell cycles in fast-growing cells. Commitment of MF cell cycles was induced by a cell permeant cADPR agonist, but blocked by a specific antagonist of cADPR-induced Ca(2+) release. Our results establish cADPR as a link between cellular metabolism and cell cycle control.

Enzymatic utilization of nicotinamide adenine dinucleotide (NAD) has increasingly been shown to have fundamental roles in gene regulation, signal transduction, and protein modification. Many of the processes require the cleavage of the nicotinamide moiety from the substrate and the formation of a reactive intermediate. Using X-ray crystallography, we show that human CD38, an NAD-utilizing enzyme, is capable of catalyzing the cleavage reactions through both covalent and noncovalent intermediates, depending on the substrate used. The covalent intermediate is resistant to further attack by nucleophiles, resulting in mechanism-based enzyme inactivation. The noncovalent intermediate is stabilized mainly through H-bond interactions, but appears to remain reactive. Our structural results favor the proposal of a noncovalent intermediate during normal enzymatic utilization of NAD by human CD38 and provide structural insights into the design of covalent and noncovalent inhibitors targeting NAD-utilization pathways.