Summary SARM1, an NAD-utilizing enzyme, regulates axonal degeneration. We show that CZ-48, a cell-permeant mimetic of NMN, activated SARM1 in vitro and in cellulo to cyclize NAD and produce a Ca 2+ messenger, cADPR, with similar efficiency as NMN. Knockout of NMN-adenylyltransferase elevated cellular NMN and activated SARM1 to produce cADPR, confirming NMN was its endogenous activator. Determinants for the activating effects and cell permeability of CZ-48 were identified. CZ-48 activated SARM1 via a conformational change of the auto-inhibitory domain and dimerization of its catalytic domain. SARM1 catalysis was similar to CD38, despite having no sequence similarity. Both catalyzed similar set of reactions, but SARM1 had much higher NAD-cyclizing activity, making it more efficient in elevating cADPR. CZ-48 acted selectively, activating SARM1 but inhibiting CD38. In SARM1-overexpressing cells, CZ-48 elevated cADPR, depleted NAD and ATP, and induced non-apoptotic death. CZ-48 is a specific modulator of SARM1 functions in cells.

CD38, as a cell surface antigen is highly expressed in several hematologic malignancies including multiple myeloma (MM) and has been proven to be a good target for immunotherapy of the disease. CD38 is also a signaling enzyme responsible for the metabolism of two novel calcium messenger molecules. To be able to target this multifunctional protein, we generated a series of nanobodies against CD38 with high affinities. Crystal structures of the complexes of CD38 with the nanobodies were solved, identifying three separate epitopes on the carboxyl domain. Chromobodies, engineered by tagging the nanobody with fluorescence proteins, provide fast, simple and versatile tools for quantifying CD38 expression. Results confirmed that CD38 was highly expressed in malignant MM cells compared with normal white blood cells. The immunotoxin constructed by splicing the nanobody with a bacterial toxin, PE38 shows highly selective cytotoxicity against patient-derived MM cells as well as the cell lines, with half maximal effective concentration reaching as low as 10−11 molar. The effectiveness of the immunotoxin can be further increased by stimulating CD38 expression using retinoid acid. These results set the stage for the development of clinical therapeutics as well as diagnostic screening for myeloma.

CD38 catalyzes the synthesis of the Ca 2+ messenger, cyclic ADP-ribose (cADPR). It is generally considered to be a type II protein with the catalytic domain facing outside. How it can catalyze the synthesis of intracellular cADPR that targets the endoplasmic Ca 2+ stores has not been resolved. We have proposed that CD38 can also exist in an opposite type III orientation with its catalytic domain facing the cytosol. Here, we developed a method using specific nanobodies to immunotarget two different epitopes simultaneously on the catalytic domain of the type III CD38 and firmly established that it is naturally occurring in human multiple myeloma cells. Because type III CD38 is topologically amenable to cytosolic regulation, we used yeast-two-hybrid screening to identify cytosolic Ca 2+ and integrinbinding protein 1 (CIB1), as its interacting partner. The results from immunoprecipitation, ELISA, and bimolecular fluorescence complementation confirmed that CIB1 binds specifically to the catalytic domain of CD38, in vivo and in vitro. Mutational studies established that the N terminus of CIB1 is the interacting domain. Using shRNA to knock down and Cas9/guide RNA to knock out CIB1, a direct correlation between the cellular cADPR and CIB1 levels was demonstrated. The results indicate that the type III CD38 is functionally active in producing cellular cADPR and that the activity is specifically modulated through interaction with cytosolic CIB1.CD38 | cyclic ADP-ribose | membrane topology | calcium signaling | CIB1 C yclic ADP-ribose (cADPR) is a second-messenger molecule regulating the endoplasmic Ca 2+ stores in a wide range of cells spanning three biological kingdoms (1-4). Equally widespread is the enzyme activity that cyclizes nicotinamide-adenine dinucleotide (NAD) to produce cADPR (5). The first fully characterized enzyme was the ADP-ribosyl cyclase, a small soluble protein in Aplysia (6). It was thus surprising that the soluble cyclase shows sequence homology with the carboxyl (C-) domain of a mammalian membrane protein, CD38, a surface antigen first identified in lymphocytes (7). Subsequent work shows that CD38 is indeed a novel enzyme catalyzing not only the synthesis of cADPR but its hydrolysis to ADP-ribose as well (8,9). Gene ablation studies establish that CD38 is the dominant enzyme in mammalian tissues responsible for metabolizing cADPR and is important in regulating diverse physiological functions, ranging from neutrophil chemotaxis to oxytocin release and insulin secretion (10-12).It is generally believed that CD38 is a type II protein with a single transmembrane segment and a short amino(N)-tail of 21 residues extending into the cytosol, whereas the major portion of the molecule, its catalytic C-domain, is facing outside (13). This membrane topology seems paradoxical to CD38 functioning as a signaling enzyme producing an intracellular messenger. A vesicular mechanism has been proposed, positing that CD38 can be endocytosed, together with transport proteins, into endolysosomes. The transporters can then...

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