Resolving the topological enigma in Ca2+ signaling by cyclic ADP-ribose and NAADP

Abstract

Cyclic ADP-ribose (cADPR) and nicotinic acid adenine dinucleotide phosphate (NAADP) are two structurally distinct messengers that mobilize the endoplasmic and endolysosomal Ca²⁺ stores, respectively. Both are synthesized by the CD38 molecule (CD38), which has long been thought to be a type II membrane protein whose catalytic domain, intriguingly, faces to the outside of the cell. Accordingly, for more than 20 years, it has remained unresolved how CD38 can use cytosolic substrates such as NAD and NADP to produce messengers that target intracellular Ca²⁺ stores. The discovery of type III CD38, whose catalytic domain faces the cytosol, has now begun to clarify this topological conundrum. This article reviews the ideas and clues leading to the discovery of the type III CD38; highlights an innovative approach for uncovering its natural existence; and discusses the regulators of its activity, folding, and degradation. We also review the compartmentalization of cADPR and NAADP biogenesis. We further discuss the possible mechanisms that promote type III CD38 expression and appraise a proposal of a Ca²⁺-signaling mechanism based on substrate limitation and product translocation. The surprising finding of another enzyme that produces cADPR and NAADP, sterile α and TIR motif-containing 1 (SARM1), is described. SARM1 regulates axonal degeneration and has no sequence similarity with CD38 but can catalyze the same set of multireactions and has the same cytosolic orientation as the type III CD38. The intriguing finding that SARM1 is activated by nicotinamide mononucleotide to produce cADPR and NAADP suggests that it may function as a regulated Ca²⁺-signaling enzyme like CD38.

Keywords: CD38; SARM1; calcium intracellular release; calcium signaling; cyclic ADP ribose (cADPR); nicotinic acid adenine dinucleotide phosphate (NAADP); protein topology; signal transduction.

Figure 1.

Type II and III CD38. The structure of the catalytic C-domain is based on crystallography (PDB entry 1YH3) and is shown with NAD at the active site. Its six disulfides are indicated and colored cyan. The N-terminal side of the transmembrane segment (orange helix) of type II CD38 has three positive arginines (red R), one more than the C-terminal side. Changing these positive residues to aspartates (green D) converts type II to type III with the catalytic domain facing the cytosol. CIB1 (PDB entry 1XO5) has four Ca²⁺-binding sites, and it interacts with the cytosolic C-domain of the type III CD38.

Figure 2.

Immunoassay and DepID for detecting type III CD38 inside cells. The immunoassay uses externally applied antibody against the N-terminal tail to detect the tail of the type III CD38 exposed to the outside. DepID probes are constructed with Nbs (blue and cyan) against two different epitopes on the catalytic domain of type III CD38, and each is fused with a luciferase fragment (LucN and LucC, semicircle). Binding of the Nbs to the epitopes on the type III CD38 (PDB entry 3RAJ) brings the luciferase fragment together, reconstituting the luciferase and producing luminescence (light green).

Figure 3.

Regulators of the type III CD38. A, CIB1 interacts with type III CD38 and modulates its cADPR-producing activity. The correct folding and disulfide formation of type III CD38 are assisted by chaperones Hsp90 and ST13. On the other hand, chaperones Hsc70, DnaJA1, and DnaJA2 mediate degradation of type III CD38 via the lysosomal receptor Lamp2A. B, Nox4 and its associated component p22^phox activate mouse type III CD38 (PDB entry 2EG9) by oxidizing cysteine 164 to form disulfide with cysteine 177.

Figure 4.

Potential mechanisms for expression of type III CD38. Left, translation begins in the cytosolic ribosomes. Binding of the SRPs to the signal sequence of the nascent polypeptide directs it to the translocation complex in the ER. The positive charges of the nascent polypeptide interact with the complex and result in folding of the polypeptide. Subsequent exit from the side channel of the complex results in type II insertion into the ER membrane (left). Middle, kinases are known to associate with the ribosomes, and the phosphorylation of the nascent polypeptide can reduce its charge interaction with the translocation complex and thus the folding of the polypeptide, resulting in the type III orientation. The lipid content of the ER can also modulate the membrane orientation of CD38, as is observed in bacteria. Right, chaperones such as Hsp70 or Ssa1 can bind to the hydrophobic segment of the nascent polypeptide and, through the Sec63 complex, direct its insertion into the ER membrane in the type III orientation via the translocation complex.

Figure 5.

The CD38/cADPR/NAADP-signaling pathway. Type III CD38, present in the ER and plasma membrane, cyclizes cytosolic NAD to produce cellular cADPR (PDB entry 2O3Q), which targets the ryanodine receptor in the ER and releases Ca²⁺ from the store. The activity of the type III CD38 is modulated by CIB1, a Ca²⁺-binding regulator. Type II CD38 is expressed on the cell surface and can be internalized through endocytosis to the endolysosomes, where the acidic lumen is conducive for NAADP production. Transporters internalized together with the type II CD38 facilitate the movement of NAADP (PDB entry 4F45) into the cytosol, where it targets Ca²⁺ channels, such as the two-pore channel, in the acidic Ca²⁺ stores. CZ48 (PDB entry 3ROM) is a mimetic of NMN and is cell-permeant. It induces dimerization and conformational changes in SARM1, releasing the catalytic TIR domain from the autoinhibition of the ARM domain. The amino segment of SARM1 is associated with the mitochondria, whereas the TIR is facing the cytosol. The activated SARM1 cyclizes NAD to produce cADPR in a manner similar to CD38.