Connie Mo Ching Lam

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.

Design, synthesis and biological characterization of novel inhibitors of CD38.

Human CD38 is a novel multi-functional protein that acts not only as an antigen for B-lymphocyte activation, but also as an enzyme catalyzing the synthesis of a Ca(2+) messenger molecule, cyclic ADP-ribose, from NAD(+). It is well established that this novel Ca(2+) signaling enzyme is responsible for regulating a wide range of physiological functions. Based on the crystal structure of the CD38/NAD(+) complex, we synthesized a series of simplified N-substituted nicotinamide derivatives (Compound 1-14). A number of these compounds exhibited moderate inhibition of the NAD(+) utilizing activity of CD38, with Compound 4 showing the highest potency. The crystal structure of CD38/Compound 4 complex and computer simulation of Compound 7 docking to CD38 show a significant role of the nicotinamide moiety and the distal aromatic group of the compounds for substrate recognition by the active site of CD38. Biologically, we showed that both Compounds 4 and 7 effectively relaxed the agonist-induced contraction of muscle preparations from rats and guinea pigs. This study is a rational design of inhibitors for CD38 that exhibit important physiological effects, and can serve as a model for future drug development.

Design, synthesis and biological evaluation of noncovalent inhibitors of human CD38 NADase.

Human CD38 is a type II transmembrane glycoprotein originally identified as a lymphocyte differentiation antigen. It has now been established that it is not lymphocyte specific, but is ubiquitously expressed in virtually all mammalian tissues examined. As a multifunctional enzyme and a member of the ADPribosyl cyclase family, CD38 catalyzes the transformation of nicotinamide adenine dinucleotide (NAD) and nicotinamide adenine dinucleotide phosphate (NADP) into two distinct Ca + messengers: cyclic ADP-ribose (cADPR) and nicotinic acid adenine dinucleotide phosphate (NAADP). CD38 can also hydrolyze cADPR and NAD to produce another Ca messenger: ADP-ribose (ADPR). 7] Both cADPR and NAADP mediate mobilization of intracellular Ca stores, targeting the endoplasmic and lysosomal stores, respectively, while ADPR targets a Ca + influx channel, TRPM2. Thus, CD38 is of vital importance for Ca signaling, which is one of the most universal and versatile processes for life. It has been proven that CD38 is directly involved in many diseases, such as diabetes, acquired immune deficiency syndrome (AIDS) 15] and chronic lymphocytic leukemia. That CD38 plays key roles in physiology provides an impetus to develop inhibitors for CD38 regulation. However, to date, only a few inhibitors of human CD38 have been reported; these compounds can be divided into two classes, covalent and noncovalent. The former includes fluoro-substituted NAD derivatives, such as arabinosyl-2’-deoxy-2’-fluoro nicotinamide adenine dinucleotide (ara-F NAD), arabinosyl-2’-deoxy-2’-fluoro nicotinamide mononucleotide (ara-F NMN), and nicotinamide 2’-deoxyriboside. The noncovalent class of inhibitors includes carbocyclic analogues of NAD, carba-NAD, and pseudocarba-NAD. 24] These compounds were shown to inhibit the NAD glycohydrolase activity of human CD38 competitively. Recently, flavonoids, for example, luteolin, were also reported to inhibit human CD38 with high potency. The covalent inhibitors, being derivatives of NAD, are susceptible to pyrophosphatase and are not able to permeate the membrane. The existing noncovalent inhibitors possess nonideal potency or selectivity. For the physiological study of human CD38, potent and structurally diverse inhibitors are needed. Since the crystal structure of CD38 was first resolved, more and more crystal structures of CD38–substrate complexes have been reported, enabling us to design noncovalent inhibitors based on this structural information. Recently, we obtained a hit compound (S125; 1) from the Sigma and Maybridge libraries by using a computer-aided virtual screening approach (Figure 1). S125 (1) exhibits an IC50 value of 86 mm against human CD38 NADase. Given the potency of this hit compound, a structural optimization and structure– activity relationship (SAR) study was carried, and the results of this study are described herein.

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.

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.

cAMP IN THE CELL CYCLE OF THE DINOFLAGELLATE CRYPTHECODINIUM COHNII (DINOPHYTA)

The second messenger cAMP is a key regulator of growth in many cells. Previous studies showed that cAMP could reverse the growth inhibition of indoleamines in the dinoflagellate Crypthecodinium cohnii Biecheler. In the present study, we measured the level of intracellular cAMP during the cell cycle of C. cohnii. cAMP peaked during the G1 phase and decreased to a minimum during S phase. Similarly, cAMP‐dependent protein kinase activities peaked at both G1 and G2+M phases of the cell cycle, decreasing to a minimum at S phase. Addition of N6, O2′‐dibutyryl (Bt2)‐cAMP directly stimulated the growth of C. cohnii. Flow cytometric analysis of synchronized C. cohnii cells suggested that 1 mM cAMP shortened the cell cycle, probably at the exit from mitosis. The size of Bt2‐cAMP treated cells at G1 was also larger than the control cells. The present study demonstrated a regulatory role of cAMP in the cell cycle progression in dinoflagellates.

Cyclic ADP-ribose links metabolism to multiple fission in the dinoflagellate Crypthecodinium cohnii

Cellular metabolism is required for cell proliferation. However, the way in which metabolic signals are conveyed to cell cycle decisions is unclear. Cyclic ADP-ribose (cADPR), the NAD(+) metabolite, mobilizes calcium from calcium stores in many cells. We found that dinoflagellate cells with higher metabolic rate underwent multiple fission (MF), a division mode in which cells can exceed twice their sizes at G1. A temperature shift-down experiment suggested that MF involves a commitment point at late G1. In fast-growing cells, cADPR level peaked in G(1) and increased with increasing concentrations of glucose in the medium. Addition of glycolytic poison iodoacetate inhibited cell growth, reduced cADPR levels as well as the commitment of cell cycles in fast-growing cells. Commitment of MF cell cycles was induced by a cell permeant cADPR agonist, but blocked by a specific antagonist of cADPR-induced Ca(2+) release. Our results establish cADPR as a link between cellular metabolism and cell cycle control.

A dinoflagellate mutant with higher frequency of multiple fission

SummaryThe dinoflagellateCrypthecodinium cohnii Biecheler propagates by both binary and multiple fission. By a newly developed mutagenesis protocol based on using ethyl methanesulfonate and a cell size screening method, a cell cycle mutant,mƒ2, was isolated with giant cells which predominantly divide by multiple fission. The average cell size of the mutantmƒ2 is larger than the controlC. cohnii. Cell cycle synchronization experiments suggest that mutantmƒ2, when compared with the control strain, has a prolonged G1 phase with a corresponding delay of the G2+M phase.

Flow cytometric analysis of nocodazole-induced cell-cycle arrest in the pennate diatom Phaeodactylum tricornutum Bohlin

The microtubule inhibitor, nocodazole (2.5 mg L-1), can arrest the cell cycle of the pennate diatom Phaeodactylum tricornutum Bohlin at G2 + M phase. Flow cytometric analysis of cells treated with nocodazole suggest that the proportion of cells at G2 + M phase can accumulate to over 95%. Even after a 48-h treatment with nocodazole (2.5 mg L-1), the cells can still exit mitosis, suggesting that the cell-cycle arrest is reversible.