Cyclic ADP-ribose is believed to be an important calcium-mobilizing second messenger in invertebrate, mammalian and plant cells. CD38, the best-characterized mammalian ADP-ribosyl cyclase, is postulated to be an important source of cyclic ADP-ribose in vivo. Using CD38-deficient mice, we demonstrate that the loss of CD38 renders mice susceptible to bacterial infections due to an inability of CD38-deficient neutrophils to directionally migrate to the site of infection. Furthermore, we show that cyclic ADP-ribose can directly induce intracellular Ca++ release in neutrophils and is required for sustained extracellular Ca++ influx in neutrophils that have been stimulated by the bacterial chemoattractant, formyl-methionyl-leucyl-phenylalanine (fMLP). Finally, we demonstrate that neutrophil chemotaxis to fMLP is dependent on Ca++ mobilization mediated by cyclic ADP-ribose. Thus, CD38 controls neutrophil chemotaxis to bacterial chemoattractants through its production of cyclic ADP-ribose, and acts as a critical regulator of inflammation and innate immune responses.

Silent information regulator 2 (Sir2) enzymes catalyze NAD؉ -dependent protein/histone deacetylation, where the acetyl group from the lysine ⑀-amino group is transferred to the ADP-ribose moiety of NAD ؉ , producing nicotinamide and the novel metabolite O-acetyl-ADPribose. Sir2 proteins have been shown to regulate gene silencing, metabolic enzymes, and life span. Recently, nicotinamide has been implicated as a direct negative regulator of cellular Sir2 function; however, the mechanism of nicotinamide inhibition was not established. Sir2 enzymes are multifunctional in that the deacetylase reaction involves the cleavage of the nicotinamide-ribosyl, cleavage of an amide bond, and transfer of the acetyl group ultimately to the 2-ribose hydroxyl of ADP-ribose. Here we demonstrate that nicotinamide inhibition is the result of nicotinamide intercepting an ADP-ribosyl-enzyme-acetyl peptide intermediate with regeneration of NAD ؉ (transglycosidation). The cellular implications are discussed. A variety of 3-substituted pyridines was found to be substrates for enzyme-catalyzed transglycosidation. A Brö nsted plot of the data yielded a slope of ؉0.98, consistent with the development of a nearly full positive charge in the transition state, and with basicity of the attacking nucleophile as a strong predictor of reactivity. NAD ؉ analogues including ␤-2-deoxy-2-fluororibo-NAD ؉ and a His-to-Ala mutant were used to probe the mechanism of nicotinamide-ribosyl cleavage and acetyl group transfer. We demonstrate that nicotinamide-ribosyl cleavage is distinct from acetyl group transfer to the 2-OH ribose. The observed enzyme-catalyzed formation of a labile 1-acetylated-ADP-fluororibose intermediate using ␤-2-deoxy-2-fluororibo-NAD ؉ supports a mechanism where, after nicotinamide-ribosyl cleavage, the carbonyl oxygen of acetylated substrate attacks the C-1 ribose to form an initial iminium adduct.The acetylation state of histones is intimately coupled to transcription, DNA repair, and replication and is governed by the competing enzymatic activities of histone acetyltransferases and histone deacetylases (reviewed in Refs. 1-3). Recently a new family of histone deacetylases has emerged and is referred to as the silent information regulator 2 (Sir2) 1 family of histone/protein deacetylases (reviewed in Refs. 4 -6) or Sirtuins (7). This family is highly conserved from prokaryotes to humans (7), and there is evidence suggesting that the scope of Sir2 activity extends beyond histone deacetylation and involves other protein targets throughout the cell. In yeast, at least five Sir2-like proteins have been identified. The founding member, yeast Sir2 (ySir2), is required for all major silenced loci (reviewed in Ref. 4). A Sir2 homologue from Salmonella enterica was shown to up-regulate acetyl-CoA synthetase, through deacetylation of a critical lysine residue (8, 9). In humans, seven Sir2 homologues have been identified to date (7). Of these seven, human SIRT2 (hSIRT2) has been identified as a cytosolic protein (10) that deacetylates ␣-tubuli...

Mice lacking CD38, an ectoenzyme that generates the calcium-mobilizing metabolite cADPR, make reduced T cell-dependent antibody responses. Despite the predicted role for CD38 in B cell activation, we find that CD38 regulates the migration of dendritic cell (DC) precursors from the blood to peripheral sites and controls the migration of mature DCs from sites of inflammation to lymph nodes. Thus, T cells are inefficiently primed in Cd38(-/-) mice, leading to poor humoral immune responses. We also show that CD38 and cADPR modulate calcium mobilization in chemokine-stimulated DCs and are required for the chemotaxis of immature and mature DCs to CCL2, CCL19, CCL21, and CXCL12. Therefore, CD38 regulates adaptive immunity by controlling chemokine receptor signaling in DCs.

Theory is presented for the use of secondary isotope effects to study the mechanisms of enzyme-catalyzed reactions. The actual secondary isotope effects on the various steps will be seen only when commitments are small, while if either forward or reverse commitments are larke the observed secondary isotope effect will be unity in the direction with the large commitment, and the equilibrium isotope effect in the other direction. If both commitments are large, one sees a portion of the equilibrium isotope effect determined by the ratio of forward and reverse commitments. @-Secondary isotope effects for liver alcohol dehydrogenase with cyclohexanone-2,2,6,6-d4 and yeast alcohol dehydrogenase with acetone-d6 show that hyperconjugation does not occur in the transition state, in which there apparently is little charge on the carbonyl carbon of the substrate. With lactate dehydrogenase, combination of primary and secondary (with pyruvate-d3) isotope effects allows calculation of commitments for pyruvate and lactate of 12-13 and 0.4-1.4, respectively.

Ca 2ϩ is a universal messenger from bacterial to mammalian cells since its concentration gradients across both organelle and plasma membranes can be efficiently used to communicate biological signals. Therefore, the control of the intracellular free Ca 2ϩ concentration [Ca 2ϩ ] i 3 is of crucial importance for the regulation of many cellular functions, including proliferation, contraction, fertilization, motility, apoptosis, and cell death (1). Receptor-mediated Ca 2ϩ influx from the extracellular space is one important mechanism to control [Ca 2ϩ ] i in non-excitable cells, e.g. leukocytes (2). Although the molecular machinery underlying Ca 2ϩ entry is still poorly defined, cation channels of the transient receptor potential (TRP) family that includes several subfamilies (3-5) are likely candidates for Ca 2ϩ entry pathways under the control of membrane receptors. TRPM2 (formerly LTRPC2 and TRPC7) is a member of the TRPM subfamily. TRPM2 forms non-selective Ca 2ϩ -permeable cation channels and is mainly expressed in brain and in cells of the immune system (6 -8). Opening of the channel is induced by intracellular ADP-ribose (ADPR; Refs. 6 and 7) and enhanced by increased cytosolic Ca 2ϩ (9). Whether NAD also activates TRPM2 currents is still controversial (6, 7, 10, 11). The nudix box in the cytosolic C-terminal region of TRPM2, a conserved motif of enzymes with nucleotide pyrophosphatase activity, seems to be responsible for gating of TRPM2 by ADPR and possibly by NAD (6,7,12). An involvement of TRPM2 in cellular signaling processes has been proposed since the expression of TRPM2 confers susceptibility to oxidant-induced cell death (11).A key question in the field relates to the potential role of the NAD metabolite ADPR as a second messenger. A function of NAD or ADPR as the missing link between specific extracellular signals and Ca 2ϩ influx mediated by TRPM2 has been hypothesized (8,13,14), but experimental proofs are missing so far. To test this hypothesis directly, we developed a method to measure intracellular levels of ADPR in Jurkat T cells (15). We report that cytosolic ADPR concentrations are raised in response to concanavalin A (ConA) and induce Ca 2ϩ entry through TRPM2, thereby significantly increasing [Ca 2ϩ ] i . Inhibition of intracellular ADPR formation or gene silencing of TRPM2 efficiently diminished receptor-mediated Ca 2ϩ influx carried by TRPM2. Moreover, blockade of ADPR formation also efficiently blocked ConA-induced cell death. EXPERIMENTAL PROCEDURESElectrophysiology-Membrane currents were recorded in the wholecell configuration of the patch clamp technique (16) or the perforatedpatch configuration with nystatin (17). An EPC9 patch clamp amplifier was used in conjunction with the PULSE stimulation and data acquisition software (HEKA Elektronik, Lamprecht, Germany). The patch electrodes were made from 1.5-mm diameter borosilicate glass capillaries and filled with intracellular solution. Data were low pass-filtered at 1 kHz and compensated for both fast and slow capacity transients. Se...