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• The Pseudomonas syringae DC3000 type III effector HopAM1 suppresses plant immunity and contains a Toll/interleukin-1 receptor (TIR) domain homologous to immunity-related TIR domains of plant nucleotide-binding leucine-rich repeat receptors that hydrolyze nicotinamide adenine dinucleotide (NAD⁺) and activate immunity. In vitro and in vivo assays were conducted to determine if HopAM1 hydrolyzes NAD⁺ and if the activity is essential for HopAM1’s suppression of plant immunity and contribution to virulence. • HPLC and LC-MS were utilized to analyze metabolites produced from NAD⁺ by HopAM1 in vitro and in both yeast and plants. Agrobacterium-mediated transient expression and in planta inoculation assays were performed to determine HopAM1’s intrinsic enzymatic activity and virulence contribution. • HopAM1 is catalytically active and hydrolyzes NAD⁺ to produce nicotinamide and a novel cADPR variant (v2-cADPR). Expression of HopAM1 triggers cell death in yeast and plants dependent on the putative catalytic residue glutamic acid 191 (E191) within the TIR domain. Furthermore, HopAM1’s E191 residue is required to suppress both pattern-triggered immunity and effector-triggered immunity and promote P. syringae virulence. • HopAM1 manipulates endogenous NAD⁺ to produce v2-cADPR and promote pathogenesis. This work suggests that HopAM1’s TIR domain possesses different catalytic specificity than other TIR domain-containing NAD⁺ hydrolases and that pathogens exploit this activity to sabotage NAD⁺ metabolism for immune suppression and virulence.

CD38 is a molecule that can act as an enzyme, with NAD-depleting and intracellular signaling activity, or as a receptor with adhesive functions. CD38 can be found expressed either on the cell surface, where it may face the extracellular milieu or the cytosol, or in intracellular compartments, such as endoplasmic reticulum, nuclear membrane, and mitochondria. The main expression of CD38 is observed in hematopoietic cells, with some cell-type specific differences between mouse and human. The role of CD38 in immune cells ranges from modulating cell differentiation to effector functions during inflammation, where CD38 may regulate cell recruitment, cytokine release, and NAD availability. In line with a role in inflammation, CD38 appears to also play a critical role in inflammatory processes during autoimmunity, although whether CD38 has pathogenic or regulatory effects varies depending on the disease, immune cell, or animal model analyzed. Given the complexity of the physiology of CD38 it has been difficult to completely understand the biology of this molecule during autoimmune inflammation. In this review, we analyze current knowledge and controversies regarding the role of CD38 during inflammation and autoimmunity and novel molecular tools that may clarify current gaps in the field.

The Ca2+ and ADP ribose (ADPR)-activated cation channel TRPM2 is the closest homolog of the cold sensor TRPM8 but serves as a deep-brain warmth sensor. To unravel the molecular mechanism of heat sensing by the TRPM2 protein, we study here temperature dependence of TRPM2 currents in cell-free membrane patches across ranges of agonist concentrations. We find that channel gating remains strictly agonist-dependent even at 40°C: heating alone or in combination with just Ca2+, just ADPR, Ca2+ + cyclic ADPR, or H₂O₂ pretreatment only marginally activates TRPM2. For fully liganded TRPM2, pore opening is intrinsically endothermic, due to ~10-fold larger activation enthalpy for opening (~200 kJ/mol) than for closure (~20 kJ/mol). However, the temperature threshold is too high (>40°C) for unliganded but too low (<15°C) for fully liganded channels. Thus, warmth sensitivity around 37°C is restricted to narrow ranges of agonist concentrations. For ADPR, that range matches, but for Ca2+, it exceeds bulk cytosolic values. The supraphysiological [Ca2+] needed for TRPM2 warmth sensitivity is provided by Ca2+ entering through the channel’s pore. That positive feedback provides further strong amplification to the TRPM2 temperature response (Q10 ~ 1,000), enabling the TRPM2 protein to autonomously respond to tiny temperature fluctuations around 37°C. These functional data together with published structures suggest a molecular mechanism for opposite temperature dependences of two closely related channel proteins.

A plethora of cellular functions are controlled by calcium signals, that are greatly coordinated by calcium release from intracellular stores, the principal component of which is the sarco/endooplasmic reticulum (S/ER). In 1997 it was generally accepted that activation of various G protein-coupled receptors facilitated inositol-1,4,5-trisphosphate (IP3) production, activation of IP3 receptors and thus calcium release from S/ER. Adding to this, it was evident that S/ER resident ryanodine receptors (RyRs) could support two opposing cellular functions by delivering either highly localised calcium signals, such as calcium sparks, or by carrying propagating, global calcium waves. Coincidentally, it was reported that RyRs in mammalian cardiac myocytes might be regulated by a novel calcium mobilising messenger, cyclic adenosine diphosphate-ribose (cADPR), that had recently been discovered by HC Lee in sea urchin eggs. A reputedly selective and competitive cADPR antagonist, 8-bromo-cADPR, had been developed and was made available to us. We used 8-bromo-cADPR to further explore our observation that S/ER calcium release via RyRs could mediate two opposing functions, namely pulmonary artery dilation and constriction, in a manner seemingly independent of IP3Rs or calcium influx pathways. Importantly, the work of others had shown that, unlike skeletal and cardiac muscles, smooth muscles might express all three RyR subtypes. If this were the case in our experimental system and cADPR played a role, then 8-bromo-cADPR would surely block one of the opposing RyR-dependent functions identified, or the other, but certainly not both. The latter seemingly implausible scenario was confirmed. How could this be, do cells hold multiple, segregated SR stores that incorporate different RyR subtypes in receipt of spatially segregated signals carried by cADPR? The pharmacological profile of 8-bromo-cADPR action supported not only this, but also indicated that intracellular calcium signals were delivered across intracellular junctions formed by the S/ER. Not just one, at least two. This article retraces the steps along this journey, from the curious pharmacological profile of 8-bromo-cADPR to the discovery of the cell-wide web, a diverse network of cytoplasmic nanocourses demarcated by S/ER nanojunctions, which direct site-specific calcium flux and may thus coordinate the full panoply of cellular processes.

Calcium (Ca2+) is a ubiquitous and fundamental signaling component that is utilized by cells to regulate a diverse range of cellular functions, such as insulin secretion from pancreatic β-cells of the islets of Langerhans. Cyclic ADP-ribose (cADPR), synthesized from NAD+ by ADP-ribosyl cyclase family proteins, such as the mammalian cluster of differentiation 38 (CD38), is important for intracellular Ca2+ mobilization for cell functioning. cADPR induces Ca2+ release from endoplasmic reticulum via the ryanodine receptor intracellular Ca2+ channel complex, in which the FK506-binding protein 12.6 works as a cADPR-binding regulatory protein. Recently, involvements of the CD38-cADPR signal system in several human diseases and animal models have been reported. This review describes the biochemical and molecular biological basis of the CD38-cADPR signal system and the diseases caused by its abnormalities.

Nicotinamide adenine dinucleotide (NAD) and its 2′-phosphorylated cousin NADP are precursors for the enzymatic formation of the Ca[sup.2+] -mobilizing second messengers adenosine diphosphoribose (ADPR), 2′-deoxy-ADPR, cyclic ADPR, and nicotinic acid adenine dinucleotide phosphate (NAADP). The enzymes involved are either NAD glycohydrolases CD38 or sterile alpha toll/interleukin receptor motif containing-1 (SARM1), or (dual) NADPH oxidases (NOX/DUOX). Enzymatic function(s) are reviewed and physiological role(s) in selected cell systems are discussed.

Although a monoclonal antibody targeting the multifunctional ectoenzyme CD38 is an FDA-approved drug, few small molecule inhibitors exist for this enzyme that catalyzes inter alia the formation and metabolism of the N1-ribosylated, Ca2+-mobilizing, second messenger cyclic adenosine 5′-diphosphoribose (cADPR). N1-Inosine 5′-monophosphate (N1-IMP) is a fragment directly related to cADPR. 8-Substituted-N1-IMP derivatives, prepared by degradation of cyclic parent compounds, inhibit CD38-mediated cADPR hydrolysis more efficiently than related cyclic analogues, making them attractive for inhibitor development. We report a total synthesis of the N1-IMP scaffold from adenine and a small initial compound series that facilitated early delineation of structure-activity parameters, with analogues evaluated for inhibition of CD38-mediated hydrolysis of cADPR. The 5′-phosphate group proved essential for useful activity, but substitution of this group by a sulfonamide bioisostere was not fruitful. 8-NH2-N1-IMP is the most potent inhibitor (IC50 = 7.6 μM) and importantly HPLC studies showed this ligand to be cleaved at high CD38 concentrations, confirming its access to the CD38 catalytic machinery and demonstrating the potential of our fragment approach.

Substantial research has demonstrated the association between cardiovascular disease and the dysregulation of intracellular calcium, ageing, reduction in nicotinamide adenine dinucleotide NAD+ content, and decrease in sirtuin activity. CD38, which comprises the soluble type, type II, and type III, is the main NADase in mammals. This molecule catalyses the production of cyclic adenosine diphosphate ribose (cADPR), nicotinic acid adenine dinucleotide phosphate (NAADP), and adenosine diphosphate ribose (ADPR), which stimulate the release of Ca2+, accompanied by NAD+ consumption and decreased sirtuin activity. Therefore, the relationship between cardiovascular disease and CD38 has been attracting increased attention. In this review, we summarize the structure, regulation, function, targeted drug development, and current research on CD38 in the cardiac context. More importantly, we provide original views about the as yet elusive mechanisms of CD38 action in certain cardiovascular disease models. Based on our review, we predict that CD38 may serve as a novel therapeutic target in cardiovascular disease in the future.

Ca2+-mobilizing adenine nucleotide second messengers cyclic adenosine diphosphoribose, (cADPR), nicotinic acid adenine dinucleotide phosphate (NAADP), adenosine diphosphoribose (ADPR), and 2′deoxy-ADPR were discovered since the late 1980s. They either release Ca2+ from endogenous Ca2+ stores, e.g., endoplasmic reticulum or acidic organelles, or evoke Ca2+ entry by directly activating a Ca2+ channel in the plasma membrane. For 25 years, Professor Barry Potter has been one of the major medicinal chemists in this topical area, designing and contributing numerous analogues to develop structure–activity relationships (SAR) as a basis for tool development in biochemistry and cell biology and for lead development in proof-of-concept studies in disease models. With this review, I wish to acknowledge our 25-year-long collaboration on Ca2+-mobilizing adenine nucleotide second messengers as a major part of Professor Potter’s scientific lifetime achievements on the occasion of his retirement in 2020.

TRPM2 is a non-selective, Ca -permeable cation channel widely expressed in immune cells. It is firmly established that the channel can be activated by intracellular adenosine 5'-diphosphoribose (ADPR). Until recent cryo-EM structures have exhibited an additional nucleotide binding site in the N-terminus of the channel, this activation was thought to occur via binding to a C-terminal domain of the channel that is highly homologous to the ADPR pyrophosphatase NudT9. Over the years it has been controversially discussed whether the Ca mobilizing second messenger cyclic ADP ribose (cADPR) might also directly activate Ca entry via TRPM2. Here we will review the status of this discussion.