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Abstract: Cyclic adenosine diphosphate ribose (cADPR) is a naturally occurring cyclic nucleotide and represents a novel class of endogenous Ca2+ messengers implicated in the regulation of the gating properties of ryanodine receptors (RyRs). This action of cADPR occurs independently from the inositol-1,4,5-trisphosphate (IP3) receptor. The regulation of intracellular Ca2+ release is a fundamental element of cellular Ca2+ homeostasis since a number of smooth muscle functions (tone, proliferation, apoptosis, and gene expression) are modulated by intracellular Ca2+ concentration ([Ca2+]i). There has been a surge in the efforts aimed at understanding the mechanisms of cADPR-mediated Ca2+ mobilization and its impact on smooth muscle function. This review summarizes the proposed roles of cADPR in the regulation of smooth muscle tone. [Copyright &y& Elsevier]

Presents information on a study which explored the existence of calcium&sup2+; messengers, cyclic adenosine diphosphate (ADP)-ribose and nicotinic acid adenine dinucleotide phosphate, in cells. Structures and mechanisms of ADP-ribosyl cyclase and CD38; Regulation of insulin secretion in pancreatic Β-cells; Potentiation of calcium&sup2+;-induced calcium&sup2+;-release and transmitter release in neuron.

Presents information on a study which examined the mechanisms for transducing external signals to intracellular responses by the molecules ADP-ribose and nicotinic acid adenine dinucleotide phosphate (NAADP). Methodology used in experiments; What are the membrane preparations of these molecules; Details on the findings of the study.

Presents the role of dual receptors in calcium mobilization during the fertilization of sea urchin eggs. Calcium mobilizing systems activated during fertilization; Binding of inositol trisphosphate and cyclic adenosine diphosphate-ribose in mobilizing calcium; Physiological role of cyclic adenosine diphosphate-ribose in calcium signaling process.

Demonstrates the modulation of cyclic adenosine diphosphate ribose (cADPR) activation of the ryanodine receptor by calmodulin. Percoll gradient purified sea urchin egg microsomes' loss of sensitivity to cADPR; Restoration of the response by a soluble protein in the supernatant; Sensitization of the calcium release mechanism.

NAADP is now established as a Ca2+ messenger by recent studies which have shown that it is active in various cells, identified the targeted Ca2+ stores and demonstrated a stimulus-activated increase in its endogenous levels. [Copyright &y& Elsevier]

A novel nanobody–drug conjugate (NDC) was constructed by incorporating an amphipathic peptide, GALA, which improved the cytotoxicity by one to two orders of magnitude. Mechanistic studies demonstrate that tethering to lipids induces GALA to form a helix, which dramatically enhances endocytosis. Our work provides a general strategy not only for improving the anti-cancer efficacy of protein-drug conjugates but also for increasing the efficiency of other types of endocytosis-dependent cell delivery. [ABSTRACT FROM AUTHOR]

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 D ual e pitopes p rotein ID entification (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. [ABSTRACT FROM AUTHOR]

CD38 catalyzes the synthesis of two structurally distinct messengers for Ca 2 + -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 Ca 2 + -signaling. This article is part of a Special Issue entitled: 13th European Symposium on Calcium. [ABSTRACT FROM AUTHOR]

Few inhibitors exist for CD38, a multifunctional enzyme catalyzing the formation and metabolism of the Ca2+-mobilizing second messenger cyclic adenosine 5′-diphosphoribose (cADPR). Synthetic, non-hydrolyzable ligands can facilitate structure-based inhibitor design. Molecular docking was used to reproduce the crystallographic binding mode of cyclic inosine 5′-diphosphoribose (N1-cIDPR) with CD38, revealing an exploitable pocket and predicting the potential to introduce an extra hydrogen bond interaction with Asp-155. The purine C-8 position of N1-cIDPR (IC50 276 µM) was extended with an amino or diaminobutane group and the 8-modified compounds were evaluated against CD38-catalyzed cADPR hydrolysis. Crystallography of an 8-amino N1-cIDPR:CD38 complex confirmed the predicted interaction with Asp-155, together with a second H-bond from a realigned Glu-146, rationalizing the improved inhibition (IC50 56 µM). Crystallography of a complex of cyclic ADP-carbocyclic ribose (cADPcR, IC50 129 µM) with CD38 illustrated that Glu-146 hydrogen bonds with the ligand N6-amino group. Both 8-amino N1-cIDPR and cADPcR bind deep in the active site reaching the catalytic residue Glu-226, and mimicking the likely location of cADPR during catalysis. Substantial overlap of the N1-cIDPR “northern” ribose monophosphate and the cADPcR carbocyclic ribose monophosphate regions suggests that this area is crucial for inhibitor design, leading to a new compound series of N1-inosine 5′-monophosphates (N1-IMPs). These small fragments inhibit hydrolysis of cADPR more efficiently than the parent cyclic compounds, with the best in the series demonstrating potent inhibition (IC50 = 7.6 µM). The lower molecular weight and relative simplicity of these compounds compared to cADPR make them attractive as a starting point for further inhibitor design. [ABSTRACT FROM AUTHOR]

Abstract: Protein ADP-ribosylation, including mono- and poly-ADP-ribosylation, is increasingly recognized to play important roles in various biological pathways. Molecular understanding of the functions of ADP-ribosylation requires the identification of the sites of modification. Although tandem mass spectrometry (MS/MS) is widely recognized as an effective means for determining protein modifications, identification of ADP-ribosylation sites has been challenging due to the labile and hydrophilic nature of the modification. Here we applied precursor ion scanning-triggered MS/MS analysis on a hybrid quadrupole linear ion trap mass spectrometer for selectively detecting ADP-ribosylated peptides and determining the auto-ADP-ribosylation sites of CD38 (cluster of differentiation 38) E226D and E226Q mutants. CD38 is an enzyme that catalyzes the hydrolysis of nicotinamide adenine dinucleotide (NAD) to ADP-ribose. Here we show that NAD can covalently label CD38 E226D and E226Q mutants but not wild-type CD38. In this study, we have successfully identified the D226/Q226 and K129 residues of the two CD38 mutants being the ADP-ribosylation sites using precursor ion scanning hybrid quadrupole linear ion trap mass spectrometry. The results offer insights about the CD38 enzymatic reaction mechanism. The precursor ion scanning method should be useful for identifying the modification sites of other ADP-ribosyltransferases such as poly(ADP-ribose) polymerases. [Copyright &y& Elsevier]

Abstract: Cyclic ADP-ribose (cADPR) is a calcium messenger that can mobilize intracellular Ca2+ stores and activate Ca2+ influx to regulate a wide range of physiological processes. Aplysia cyclase is the first member of the ADP-ribosyl cyclases identified to catalyze the cyclization of NAD+ into cADPR. The catalysis involves a two-step reaction, the elimination of the nicotinamide ring and the cyclization of the intermediate resulting in the covalent attachment of the purine ring to the terminal ribose. Aplysia cyclase exhibits a high degree of leniency towards the purine base of its substrate, and the cyclization reaction takes place at either the N1- or the N7-position of the purine ring. To decipher the mechanism of cyclization in Aplysia cyclase, we used a crystallization setup with multiple Aplysia cyclase molecules present in the asymmetric unit. With the use of natural substrates and analogs, not only were we able to capture multiple snapshots during enzyme catalysis resulting in either N1 or N7 linkage of the purine ring to the terminal ribose, we were also able to observe, for the first time, the cyclized products of both N1 and N7 cyclization bound in the active site of Aplysia cyclase. [Copyright &y& Elsevier]

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. [ABSTRACT FROM AUTHOR]

Summary: Cyclic ADP-ribose is an important Ca2+-mobilizing cytosolic messenger synthesized from β-NAD+ by ADP-ribosyl cyclases (ARCs). However, the focus upon ectocellular mammalian ARCs (CD38 and CD157) has led to confusion as to how extracellular enzymes generate intracellular messengers in response to stimuli. We have cloned and characterized three ARCs in the sea urchin egg and found that endogenous ARCβ and ARCγ are intracellular and located within the lumen of acidic, exocytotic vesicles, where they are optimally active. Intraorganelle ARCs are shielded from cytosolic substrate and targets by the organelle membrane, but this barrier is circumvented by nucleotide transport. We show that a β-NAD+ transporter provides ARC substrate that is converted luminally to cADPR, which, in turn, is shuttled out to the cytosol via a separate cADPR transporter. Moreover, nucleotide transport is integral to ARC activity physiologically because three transport inhibitors all inhibited the fertilization-induced Ca2+ wave that is dependent upon cADPR. This represents a novel signaling mechanism whereby an extracellular stimulus increases the concentration of a second messenger by promoting messenger transport from intraorganelle synthesis sites to the cytosol. [Copyright &y& Elsevier]

Summary: Enzymatic utilization of nicotinamide adenine dinucleotide (NAD) has increasingly been shown to have fundamental roles in gene regulation, signal transduction, and protein modification. Many of the processes require the cleavage of the nicotinamide moiety from the substrate and the formation of a reactive intermediate. Using X-ray crystallography, we show that human CD38, an NAD-utilizing enzyme, is capable of catalyzing the cleavage reactions through both covalent and noncovalent intermediates, depending on the substrate used. The covalent intermediate is resistant to further attack by nucleophiles, resulting in mechanism-based enzyme inactivation. The noncovalent intermediate is stabilized mainly through H-bond interactions, but appears to remain reactive. Our structural results favor the proposal of a noncovalent intermediate during normal enzymatic utilization of NAD by human CD38 and provide structural insights into the design of covalent and noncovalent inhibitors targeting NAD-utilization pathways. [Copyright &y& Elsevier]

Summary: Human CD38 is a multifunctional protein involved in diverse functions. As an enzyme, it is responsible for the synthesis of two Ca2+ messengers, cADPR and NAADP; as an antigen, it is involved in regulating cell adhesion, differentiation, and proliferation. Besides, CD38 is a marker of progression of HIV-1 infection and a negative prognostic marker of B-CLL. We have determined the crystal structure of the soluble extracellular domain of human CD38 to 1.9 Å resolution. The enzyme’s overall topology is similar to the related proteins CD157 and the Aplysia ADP-ribosyl cyclase, except with large structural changes at the two termini. The extended positively charged N terminus has lateral associations with the other CD38 molecule in the crystallographic asymmetric unit. The analysis of the CD38 substrate binding models revealed two key residues that may be critical in controlling CD38’s multifunctionality of NAD hydrolysis, ADP-ribosyl cyclase, and cADPR hydrolysis activities. [Copyright &y& Elsevier]

Abstract: The cell surface antigen, CD38, is a 45-kDa transmembrane protein which is predominantly expressed on hematopoietic cells during differentiation. As a bifunctional ectoenzyme, it catalyzes the synthesis of cyclic ADP-ribose (cADPR) from NAD+ and hydrolysis of either NAD+ or cADPR to ADP-ribose. All-trans-retinoic acid (RA) is a potent and specific inducer of CD38 in myeloid cells. In this report, we demonstrate that the nuclei of RA-treated human HL-60 myeloblastic cells reveal enzymatic activities inherent to CD38. Thus, GDP-ribosyl cyclase and NAD+ glycohydrolase activities in the nuclear fraction increased very significantly in response to incubation with RA. With Western blotting, we detected in the nuclear protein fraction from RA-treated cells a ∼43-kDa protein band which was reactive with the CD38-specific monoclonal antibody OKT10. The expression of CD38 in HL-60 nuclei was also shown with FACScan analysis. RA treatment gave rise to an increase in in vitro ADP ribosylation of the ∼43-kDa nuclear protein. Moreover, nuclei isolated from RA-treated HL-60 cells revealed calcium release in response to cADPR, whereas a similar response was not observed in control nuclei. These results suggest that CD38 is expressed in HL-60 cell nuclei during RA-induced differentiation. [Copyright &y& Elsevier]

ADP-ribosyl cyclase catalyzes the elimination of nicotinamide from NAD and cyclization to cADPR, a known second messenger in cellular calcium signaling pathways. We have determined to 2.0 A˚ resolution the structure of Aplysia cyclase with ribose-5-phosphate bound covalently at C3′ and with the base exchange substrate (BES), pyridylcarbinol, bound to the active site. In addition, further refinement at 2.4 A˚ resolution of the structure of nicotinamide-bound cyclase, which was previously reported, reveals that ribose-5-phosphate is also covalently bound in this structure, and a second nicotinamide site was identified. The structures of native and mutant Glu179Ala cyclase were also solved to 1.7 and 2.0 A˚ respectively. It is proposed that the second nicotinamide site serves to promote cyclization by clearing the active site of the nicotinamide byproduct. Moreover, a ribosylation mechanism can be proposed in which the cyclization reaction proceeds through a covalently bound intermediate. [Copyright &y& Elsevier]