Abstract

It is generally believed that multiple Ca2+ stores are present in cells, a notion that has now been made substantive by the discovery of multiple Ca2+ mobilizing messengers. Cyclic ADP-ribose (cADPR) and nicotinic acid adenine dinucleotide phosphate (NAADP) are two such messengers that are derived from NAD and NADP, respectively. A wide variety of cells, from plants to mammals, including human, have been shown to be responsive to these two novel Ca2+ messengers. Not only are their structures and mechanisms of action different, their targeted Ca2+ stores are also distinct and separable. Moreover, they are derived from different substrates, cADPR from NAD and NAADP from NADP. It is thus entirely unexpected that a single enzyme is responsible for synthesizing these two different Ca2+-messengers. CD38 is one such enzyme and it belongs to a new family of homologues that includes CD157 and Aplysia ADP-ribosyl cyclase. Results using knockout mice show that CD38 is the most dominant enzyme in mammalian tissues responsible for synthesizing cADPR and NAADP. The emerging view is that cADPR and NAADP represent two branches of a novel Ca2+-signaling pathway with CD38 at its bifurcation point. Because of its pivotal role in the cADPR/NAADP-signaling pathway, it is of critical importance that the molecular mechanism of its action be elucidated. We have expressed the extra-membrane domain of CD38 in yeast as a soluble protein that retains all enzymatic activities indistinguishable from native CD38. We have mapped the active site and identified the critical residues in it by site-directed mutagenesis. These results form the basis for a unified catalytic model that accounts for the novel multiplicity of this family of signaling enzymes. We have now succeeded in crystallizing CD38 and solved its structure. The structure-function studies described in this proposal will verify and refine the catalytic model and the specific aims are: ? ? Aim 1: To determine the structural basis of the multi-catalytic functions of CD38. ? Aim 2: To determine the structural basis of regulation of CD38 catalysis. ? Aim 3: To determine the structural basis of the antigenic functions of CD38. ? Aim 4: To characterize intracellular CD38. ? ?

  Funding Agency

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
2R01GM061568-05
Application #
7028076
Study Section
Macromolecular Structure and Function C Study Section (MSFC)
Program Officer
Shapiro, Bert I
Project Start
2000-06-01
Project End
2010-01-31
Budget Start
2006-02-01
Budget End
2007-01-31
Support Year
5
Fiscal Year
2006
Total Cost
$418,545
Indirect Cost

  Institution

Name
University of Minnesota Twin Cities
Department
Pharmacology
Type
Schools of Medicine
DUNS #
555917996
City
Minneapolis
State
MN
Country
United States
Zip Code
55455

  Publications

Zhang, HongMin; Graeff, Richard; Chen, Zhe et al. (2011) Dynamic conformations of the CD38-mediated NAD cyclization captured in a single crystal. J Mol Biol 405:1070-8
Xu, Xiaodong; Graeff, Richard; Xie, Qiguang et al. (2009) Comment on ""The Arabidopsis circadian clock incorporates a cADPR-based feedback loop"". Science 326:230; author reply 230
Liu, Qun; Graeff, Richard; Kriksunov, Irina A et al. (2009) Structural basis for enzymatic evolution from a dedicated ADP-ribosyl cyclase to a multifunctional NAD hydrolase. J Biol Chem 284:27637-45
Graeff, Richard; Liu, Qun; Kriksunov, Irina A et al. (2009) Mechanism of cyclizing NAD to cyclic ADP-ribose by ADP-ribosyl cyclase and CD38. J Biol Chem 284:27629-36
Liu, Qun; Kriksunov, Irina A; Jiang, Hong et al. (2008) Covalent and noncovalent intermediates of an NAD utilizing enzyme, human CD38. Chem Biol 15:1068-78
Liu, Qun; Graeff, Richard; Kriksunov, Irina A et al. (2008) Conformational Closure of the Catalytic Site of Human CD38 Induced by Calcium. Biochemistry 47:13966-73
Liu, Qun; Kriksunov, Irina A; Wang, Zhongwu et al. (2008) Hierarchical and helical self-assembly of ADP-ribosyl cyclase into large-scale protein microtubes. J Phys Chem B 112:14682-6
Liu, Qun; Kriksunov, Irina A; Graeff, Richard et al. (2007) Structural basis for formation and hydrolysis of the calcium messenger cyclic ADP-ribose by human CD38. J Biol Chem 282:5853-61
Liu, Qun; Kriksunov, Irina A; Moreau, Christelle et al. (2007) Catalysis-associated conformational changes revealed by human CD38 complexed with a non-hydrolyzable substrate analog. J Biol Chem 282:24825-32
Graeff, Richard; Liu, Qun; Kriksunov, Irina A et al. (2006) Acidic residues at the active sites of CD38 and ADP-ribosyl cyclase determine nicotinic acid adenine dinucleotide phosphate (NAADP) synthesis and hydrolysis activities. J Biol Chem 281:28951-7

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