Yong Juan Zhao

The membrane-bound enzyme CD38 exists in two opposing orientations

The identification of two forms of a transmembrane enzyme suggests that flipping the catalytic domain from the outside to the inside of the cell may regulate its activity. Moving the Catalytic Domain to the Inside CD38 is a membrane-bound enzyme that catalyzes both the production and the hydrolysis of cyclic ADP ribose (cADPR), an intracellular messenger that stimulates the release of Ca2+ from the endoplasmic reticulum. However, CD38 is thought to exist as a type II transmembrane protein with its C-terminal catalytic domain on the outside of the cell; thus, how it contributes to intracellular signaling is controversial. With antibodies specific for the N-terminal region of CD38, Zhao et al. showed in cell lines and primary human cells the presence of a proportion of CD38 in the type III form, with the catalytic domain facing the cytosol. Mutations in the N-terminal region of CD38 flipped its orientation in the membrane so that it was only present in the type III form, and transfected cells expressing this mutant were more efficient at producing cADPR than were cells expressing wild-type CD38. Together, these data suggest that cells have CD38 molecules in opposing orientations, with the type III form predominantly responsible for signaling. The transmembrane enzyme CD38, a multifunctional protein ubiquitously present in cells, is the main enzyme that synthesizes and hydrolyzes cyclic adenosine 5′-diphosphate-ribose (cADPR), an intracellular Ca2+-mobilizing messenger. CD38 is thought to be a type II transmembrane protein with its carboxyl-terminal catalytic domain located on the outside of the cell; thus, the mechanism by which CD38 metabolizes intracellular cADPR has been controversial. We developed specific antibodies against the amino-terminal segment of CD38 and showed that two opposing orientations of CD38, type II and type III (which has its catalytic domain inside the cell), were both present on the surface of HL-60 cells during retinoic acid–induced differentiation. When activated by interferon-γ, human primary monocytes and the monocytic U937 cell line exhibited a similar co-distribution pattern. Site-directed mutagenesis experiments showed that the membrane orientation of CD38 could be converted from a mixture of type II and type III orientations to all type III by mutating the cationic amino acid residues in the amino-terminal segment of CD38. Expression of type III CD38 construct in transfected cells led to increased intracellular concentrations of cADPR, indicating the importance of the type III orientation of CD38 to its Ca2+ signaling function. The identification of these two forms of CD38 suggests that flipping the catalytic domain from the outside to the inside of the cell may be a mechanism regulating its signaling activity.

CD38/cADPR/Ca2+ pathway promotes cell proliferation and delays nerve growth factor-induced differentiation in PC12 cells.

Intracellular Ca2+ mobilization plays an important role in a wide variety of cellular processes, and multiple second messengers are responsible for mediating intracellular Ca2+ changes. Here we explored the role of one endogenous Ca2+-mobilizing nucleotide, cyclic adenosine diphosphoribose (cADPR), in the proliferation and differentiation of neurosecretory PC12 cells. We found that cADPR induced Ca2+ release in PC12 cells and that CD38 is the main ADP-ribosyl cyclase responsible for the acetylcholine (ACh)-induced cADPR production in PC12 cells. In addition, the CD38/cADPR signaling pathway is shown to be required for the ACh-induced Ca2+ increase and cell proliferation. Inhibition of the pathway, on the other hand, accelerated nerve growth factor (NGF)-induced neuronal differentiation in PC12 cells. Conversely, overexpression of CD38 increased cell proliferation but delayed NGF-induced differentiation. Our data indicate that cADPR plays a dichotomic role in regulating proliferation and neuronal differentiation of PC12 cells.

A novel fluorescent cell membrane permeable caged cyclic ADP-Ribose analogue

Background: The available agonists for cADPR, an endogenous Ca2+-mobilizing nucleotide, are either weak or not cell-permeant. Results: We synthesized a coumarin-caged isopropylidene-protected cIDPRE (Co-i-cIDPRE), which is a potent and cell-permeant cADPR agonist. Conclusion: Uncaging of Co-i-cIDPRE activates RyRs for Ca2+ mobilization and triggers Ca2+ influx via TRPM2. Significance: Co-i-cIDPRE should provide a valuable tool to study cADPR/Ca2+ signaling. Cyclic adenosine diphosphate ribose is an endogenous Ca2+ mobilizer involved in diverse cellular processes. A cell membrane-permeable cyclic adenosine diphosphate ribose analogue, cyclic inosine diphosphoribose ether (cIDPRE), can induce Ca2+ increase in intact human Jurkat T-lymphocytes. Here we synthesized a coumarin-caged analogue of cIDPRE (Co-i-cIDPRE), aiming to have a precisely temporal and spatial control of bioactive cIDPRE release inside the cell using UV uncaging. We showed that Co-i-cIDPRE accumulated inside Jurkat cells quickly and efficiently. Uncaging of Co-i-cIDPRE evoked Ca2+ release from endoplasmic reticulum, with concomitant Ca2+ influx in Jurkat cells. Ca2+ release evoked by uncaged Co-i-cIDPRE was blocked by knockdown of ryanodine receptors (RyRs) 2 and 3 in Jurkat cells. The associated Ca2+ influx, on the other hand, was abolished by double knockdown of Stim1 and TRPM2 in Jurkat cells. Furthermore, Ca2+ release or influx evoked by uncaged Co-i-cIDPRE was recapitulated in HEK293 cells that overexpress RyRs or TRPM2, respectively, but not in wild-type cells lacking these channels. In summary, our results indicate that uncaging of Co-i-cIDPRE incites Ca2+ release from endoplasmic reticulum via RyRs and triggers Ca2+ influx via TRPM2.

Determinants of the membrane orientation of a calcium signaling enzyme CD38.

CD38 catalyzes the synthesis of two structurally distinct messengers for Ca²⁺-mobilization, cyclic ADP-ribose (cADPR) and nicotinic acid adenine dinucleotide phosphate (NAADP), from cytosolic substrates, NAD and NADP, respectively. CD38 is generally thought of as a type II membrane protein with its catalytic site facing outside. We recently showed that CD38 exists, instead, in two opposite membrane orientations. The determinant for the membrane topology is unknown. Here, specific antibodies against type III CD38 were designed and produced. We show that mutating the positively charged residues in the N-terminal tail of CD38 converted its orientation to type III, with the catalytic domain facing the cytosol and it was fully active in producing intracellular cADPR. Changing the serine residues to aspartate, which is functionally equivalent to phosphorylation, had a similar effect. The mutated CD38 was expressed intracellularly and was un-glycosylated. The membrane topology could also be modulated by changing the highly conserved di-cysteine. The results indicate that the net charge of the N-terminal segment is important in determining the membrane topology of CD38 and that the type III orientation can be a functional form of CD38 for Ca²⁺-signaling. This article is part of a Special Issue entitled: 13th European Symposium on Calcium.

Immuno-targeting the multifunctional CD38 using nanobody

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.

Inhibition of NAADP signalling on reperfusion protects the heart by preventing lethal calcium oscillations via two-pore channel 1 and opening of the mitochondrial permeability transition pore

Aims In the heart, a period of ischaemia followed by reperfusion evokes powerful cytosolic Ca2+ oscillations that can cause lethal cell injury. These signals represent attractive cardioprotective targets, but the underlying mechanisms of genesis are ill-defined. Here, we investigated the role of the second messenger nicotinic acid adenine dinucleotide phosphate (NAADP), which is known in several cell types to induce Ca2+ oscillations that initiate from acidic stores such as lysosomes, likely via two-pore channels (TPCs, TPC1 and 2). Methods and results An NAADP antagonist called Ned-K was developed by rational design based on a previously existing scaffold. Ned-K suppressed Ca2+ oscillations and dramatically protected cardiomyocytes from cell death in vitro after ischaemia and reoxygenation, preventing opening of the mitochondrial permeability transition pore. Ned-K profoundly decreased infarct size in mice in vivo. Transgenic mice lacking the endo-lysosomal TPC1 were also protected from injury. Conclusion NAADP signalling plays a major role in reperfusion-induced cell death and represents a potent pathway for protection against reperfusion injury.

Design, Synthesis and SAR Studies of NAD Analogues as Potent Inhibitors towards CD38 NADase

Nicotinamide adenine dinucleotide (NAD), one of the most important coenzymes in the cells, is a substrate of the signaling enzyme CD38, by which NAD is converted to a second messenger, cyclic ADP-ribose, which releases calcium from intracellular calcium stores. Starting with 2′-deoxy-2′-fluoroarabinosyl-β-nicotinamide adenine dinucleotide (ara-F NAD), a series of NAD analogues were synthesized and their activities to inhibit CD38 NAD glycohydrolase (NADase) were evaluated. The adenosine-modified analogues showed potent inhibitory activities, among which 2′-deoxy-2′-fluoroarabinosyl-β-nicotinamideguanine dinucleotide (ara-F NGD) was the most effective one. The structure-activity relationship of NAD analogues was also discussed.

Cytosolic CD38 Protein Forms Intact Disulfides and Is Active in Elevating Intracellular Cyclic ADP-ribose

CD38 catalyzes the synthesis of cyclic ADP-ribose (cADPR), a Ca2+ messenger responsible for regulating a wide range of physiological functions. It is generally regarded as an ectoenzyme, but its intracellular localization has also been well documented. It is not known if internal CD38 is enzymatically active and contributes to the Ca2+ signaling function. In this study, we engineered a novel soluble form of CD38 that can be efficiently expressed in the cytosol and use cytosolic NAD as a substrate to produce cADPR intracellularly. The activity of the engineered CD38 could be decreased by mutating the catalytic residue Glu-226 and increased by the double mutation E146A/T221F, which increased its cADPR synthesis activity by >11-fold. Remarkably, the engineered CD38 exhibited the ability to form the critical disulfide linkages required for its enzymatic activity. This was verified by using a monoclonal antibody generated against a critical disulfide, Cys-254–Cys-275. The specificity of the antibody was established by x-ray crystallography and site-directed mutagenesis. The engineered CD38 is thus a novel example challenging the general belief that cytosolic proteins do not possess disulfides. As a further refinement of this approach, the engineered CD38 was placed under the control of tetracycline using an autoregulated construct. This study has set the stage for in vivo manipulation of cADPR metabolism.

Cyclic ADP-Ribose and Heat Regulate Oxytocin Release via CD38 and TRPM2 in the Hypothalamus during Social or Psychological Stress in Mice

Hypothalamic oxytocin (OT) is released into the brain by cyclic ADP-ribose (cADPR) with or without depolarizing stimulation. Previously, we showed that the intracellular free calcium concentration ([Ca2+]i) that seems to trigger OT release can be elevated by β-NAD+, cADPR, and ADP in mouse oxytocinergic neurons. As these β-NAD+ metabolites activate warm-sensitive TRPM2 cation channels, when the incubation temperature is increased, the [Ca2+]i in hypothalamic neurons is elevated. However, it has not been determined whether OT release is facilitated by heat in vitro or hyperthermia in vivo in combination with cADPR. Furthermore, it has not been examined whether CD38 and TRPM2 exert their functions on OT release during stress or stress-induced hyperthermia in relation to the anxiolytic roles and social behaviors of OT under stress conditions. Here, we report that OT release from the isolated hypothalami of male mice in culture was enhanced by extracellular application of cADPR or increasing the incubation temperature from 35°C to 38.5°C, and simultaneous stimulation showed a greater effect. This release was inhibited by a cADPR-dependent ryanodine receptor inhibitor and a nonspecific TRPM2 inhibitor. The facilitated release by heat and cADPR was suppressed in the hypothalamus isolated from CD38 knockout mice and CD38- or TRPM2-knockdown mice. In the course of these experiments, we noted that OT release differed markedly between individual mice under stress with group housing. That is, when male mice received cage-switch stress and eliminated due to their social subclass, significantly higher levels of OT release were found in subordinates compared with ordinates. In mice exposed to anxiety stress in an open field, the cerebrospinal fluid (CSF) OT level increased transiently at 5 min after exposure, and the rectal temperature also increased from 36.6°C to 37.8°C. OT levels in the CSF of mice with lipopolysaccharide-induced fever (+0.8°C) were higher than those of control mice. The TRPM2 mRNA levels and immunoreactivities increased in the subordinate group with cage-switch stress. These results showed that cADPR/CD38 and heat/TRPM2 are co-regulators of OT secretion and suggested that CD38 and TRPM2 are potential therapeutic targets for OT release in psychiatric diseases caused by social stress.

Alpha 1‐antichymotrypsin/SerpinA3 is a novel target of orphan nuclear receptor Nur77

Nur77 is one member of the nuclear receptor superfamily. As a transcription factor, Nur77 participates in a variety of biological processes, including T cell development, inflammatory responses, steroid hormone synthesis, and hepatic glucose metabolism. It typically acts via binding to the Nur77 responsive element (NBRE) in the promoter regions of its target genes. In the present study, we identified a novel Nur77‐regulated gene, α1‐antichymotrypsin/SerpinA3, via an approach combining computational prediction and wet‐laboratory validations. First, we identified 483 candidate genes via a human genome‐wide scan for NBREs in their proximal promoters. Three out of 14 function‐associated genes were further identified to be transactivated by Nur77 in luciferase reporter gene assays in HEK 293T cells. The transactivation assay proved that the NBRE (−182 to −175) in the SerpinA3 promoter region is a novel Nur77‐dependent functional motif in HEK 293T and HepG2 cells. Electrophoretic mobility shift and chromatin immunoprecipitation assays demonstrated that Nur77 physically associates with the SerpinA3 promoter region both in vitro and in vivo. Nur77 overexpression and RNA interference‐mediated Nur77 gene knockdown analysis confirmed that SerpinA3 is indeed a novel Nur77‐targeted gene. These data may throw light on the function of Nur77 in inflammatory responses and acute‐phase reactions as well as the development of Alzheimer’s disease.