A Cell-Permeant Mimetic of NMN Activates SARM1 to Produce Cyclic ADP-Ribose and Induce Non-apoptotic Cell Death.

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 Ca2+ 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.

Nanobody-based dual epitopes protein identification (DepID) assay for measuring soluble CD38 in plasma of multiple myeloma patients.

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.

Cytosolic interaction of type III human CD38 with CIB1 modulates cellular cyclic ADP-ribose levels.

CD38 catalyzes the synthesis of the Ca2+ 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 Ca2+ 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 Ca2+ and integrin-binding 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.