Francis Schuber

CD38: a new paradigm in lymphocyte activation and signal transduction

Summary: CD38 is a type II transmembrane glycoprotein that is extensively expressed on cells of bematopoietic and non‐hematopoietic lineage. Although the intracellular domain of CD38 is not homologous to any known proteins, the extracellular domain of CD38 is structurally related to enzymes in the ADP‐ribosyl cyclase family. The structural homology between CD 38 and die cyclase family members extends to functional homology, as the extracellular domain of CD 38 can mediate the catalysis of β‐NAD+ into nicotinamide, ADP‐ribose (ADPR) and, to a lesser extent, into cychc ADPR‐ribose (cADPR). Extensive investigation in other systems has shown tbat cADPR is an important regulator of intracellular Ca2+ release. Since engagement of CD38 on bematopoietic cells with anti‐CD38 Abs has been shown to have potent effects on a number of in vitro cellular responses, we have speculated that cADPR might control CD38‐mediated signal transduction. However, it has been difficult to understand how a mediator which is typically an intracellular signaling molecule could potentiate its effects from an extracellular location, thus posing a dilemma which pertains to all ecto‐enzymes and the mechanisms by which they regulate signal transduction and cellular processes. This review describes the biologic properties ofmurineCD38, its role in humoral immunity, and its signal transduction properties in B lymphocytes. We suggest that signaling through CD38 represents a new paradigm in lymphocyte signal transduction and is predicated upon extracellular, rather than intracellular, crosstalk.

Structure and Enzymology of ADP-ribosyl Cyclases: Conserved Enzymes that Produce Multiple Calcium Mobilizing Metabolites

Cyclic ADP-ribose is an important calcium mobilizing metabolite produced by the ADP-ribosyl cyclase (cyclases) family of enzymes. Three evolutionarily conserved ADP-ribosyl cyclase superfamily members have been identified, one from the invertebrate Aplysia californica and two from mammalian tissues, CD38 and CD157. CD38 regulates calcium signaling in a number of cell types, and it was recently shown that cyclic ADP-ribose produced by CD38 modulates calcium mobilization induced upon chemokine receptor engagement. Excitingly, because immunocytes deficient in CD38 are unable to migrate to inflammatory sites in vivo, this enzyme has now become an attractive target for drug development. To rationally design inhibitors it is critical to understand the mechanism(s) by which CD38 catalyzes the transformation of its substrate NAD+ into cyclic ADP-ribose. Likewise, it is necessary to identify the CD38 substrate-binding site. Importantly, significant progress has been made in these two areas and much is now known about the structure and enzymology of CD38 and the other ADP-ribosyl cyclase superfamily members. In this review, we will outline the critical data demonstrating a role for CD38 in regulating calcium mobilization in mammalian cells. We will also describe the crystallographic data and site-directed mutagenesis studies that have helped to elucidate the CD38 structure and the identification of its active site and key catalytic residues. Finally, we will address the important advances in our understanding of the kinetic and molecular mechanisms that control cyclic ADP-ribose production by CD38.

Biphasic induction of ornithine decarboxylase and putrescine levels in growing HTC cells.

Abstract A biphasic induction of ornithine decarboxylase with concomitant increase of intracellular putrescine was seen in growing rat hepatoma cells during each generation period. In non-growing HTC cells no coordinate accumulation of intracellular putrescine followed the unique induction of DDC by dilution into fresh serum-deprived medium. The data together suggest that the biphasic increases of ODC activity occur just before and after DNA synthesis and that a growing HTC cell has a finely regulated cycle of ODC activity. Finally, ODC activity may not always correlate with the intracellular putrescine levels.

In vitro inhibition of animal and higher plants 2,3-oxidosqualene-sterol cyclases by 2-aza-2,3- dihydrosqualene and derivatives, and by other ammonium-containing molecules

2-Aza-2,3-dihydrosqualene and related molecules, a series of new compounds designed as analogues of the transient carbocationic high energy intermediate, occurring in the oxirane ring opening during the cyclization of 2,3-oxidosqualene, were tested in vitro as inhibitors of the microsomal 2,3-oxidosqualene cyclase of animals (rat liver) and of higher plants (maize, pea). These molecules proved to be good and specific inhibitors for the cyclases of both phyla. The inhibition is due to positively charged species and is sensitive to the steric hindrance around the nitrogen-atom. 4,4,10 beta-Trimethyl-trans-decal-3 beta-ol and 4,10 beta-dimethyl-trans-decal-3 beta-ol, which have previously been described (J.A. Nelson et al., J. Am. chem. Soc. 100, 4900 (1978] as inhibitors of the 2,3-oxidosqualene cyclase of chinese hamster ovary cells, were found to be non-competitive inhibitors of the rat liver microsomal enzyme and presented no activity towards the higher plants cyclases. Aza derivatives of these decalines (A. Rahier et al., Phytochemistry, in press), which were aimed to mimic the C-8 carbocationic intermediate occurring during later steps of the 2,3-oxidosqualene cyclization did not inhibit the cyclases. This result underlines the theoretical limitations of the high energy analogues concept in designing enzyme inhibitors. Amongst other molecules tested, 2,3-epiminosqualene was found to be a reversible, non-competitive inhibitor of the cyclases; similarly U18666A was a very potent inhibitor of the microsomal cyclases. In contrast AMO 1618, a known anticholesterolemic agent reported previously to act at the level of the 2,3-oxidosqualene cyclization step, was not found per se to act on the cyclases.

Evidence for an Intracellular ADP-ribosyl Cyclase/NAD+-glycohydrolase in Brain from CD38-deficient Mice

Cyclic ADP-ribose, a metabolite of NAD+, is known to modulate intracellular calcium levels and signaling in various cell types, including neural cells. The enzymes responsible for producing cyclic ADP-ribose in the cytoplasm of mammalian cells remain unknown; however, two mammalian enzymes that are capable of producing cyclic ADP-ribose extracellularly have been identified, CD38 and CD157. The present study investigated whether an ADP-ribosyl cyclase/NAD+-glycohydrolase independent of CD38 is present in brain tissue. To address this question, NAD+ metabolizing activities were accurately examined in developing and adult Cd38-/- mouse brain protein extracts and cells. Low ADP-ribosyl cyclase and NAD+-glycohydrolase activities (in the range of pmol of product formed/mg of protein/min) were detected in Cd38-/- brain at all developmental stages studied. Both activities were found to be associated with cell membranes. The activities were significantly higher in Triton X-100-treated neural cells compared with intact cells, suggesting an intracellular location of the novel cyclase. The cyclase and glycohydrolase activities were optimal at pH 6.0 and were inhibited by zinc, properties which are distinct from those of CD157. Both activities were enhanced by guanosine 5′-O-(3-thiotriphosphate), a result suggesting that the novel enzyme may be regulated by a G protein-dependent mechanism. Altogether our results indicate the presence of an intracellular membrane-bound ADP-ribosyl cyclase/NAD+-glycohydrolase distinct from CD38 and from CD157 in mouse brain. This novel enzyme, which is more active in the developing brain than in the adult tissue, may play an important role in cyclic ADP-ribose-mediated calcium signaling during brain development as well as in adult tissue.

Immunogenicity of new heterobifunctional cross-linking reagents used in the conjugation of synthetic peptides to liposomes

We have investigated the immunogenicity of six thiol-reactive heterobifunctional cross-linking reagents that permit the conjugation of cysteine carrying peptides to the surface of liposome containing monophosphoryl lipid A. Such constructs elicit an immune response against short synthetic peptides and our aim was to find the least immunogenic linkers to limit potential carrier-induced epitopic suppression. For that purpose the properties of three new polyoxyethylene linkers of different lengths and thiol-reactive moieties (maleimide, bromoacetyl, dithiopyridine) were compared to known derivatives obtained by reacting the classical reagents SMPB and SPDP or N-succinimidyl bromoacetate with phosphatidylethanolamine. The least immunogenic linkers were the bromoacetate derivatives whereas those containing a maleimide group evoked a significant anti-linker immune response. In addition, using IRGERA as a model peptide, we found that all six liposomal constructs strongly elicited the production of anti-peptide IgG antibodies. This immune response was therefore independent of the length of the linkers (ranging between 0.3 and 1.6 nm) and of the nature of the linkage. between the peptide and the thiol-reactive moieties of the cross-linkers, i.e. stable thioether or bio-reducible disulfide bonds.

Antitumor activity of liposomal ErbB2/HER2 epitope peptide-based vaccine constructs incorporating TLR agonists and mannose receptor targeting

Synthetic and molecularly defined constructs containing the minimal components to mimic and amplify the physiological immune response are able to induce an efficient cytotoxic response. In the current study this approach was applied to the development of highly versatile liposomal constructs to co-deliver peptide epitopes in combination with TLR agonists in order to induce a specific anti-tumor cellular immune response against ErbB2 protein-expressing tumor cells. Liposomes containing ErbB2 p63-71 cytotoxic T lymphocyte (CTL) and HA307-319 T- helper (Th) peptide epitopes associated to innovative synthetic TLR2/1 (Pam(3)CAG) or TLR2/6 agonists (Pam(2)CAG and Pam(2)CGD), were injected in mice bearing ErbB2 protein-expressing tumor cells. Mannosylated ligands were also incorporated into the constructs to target antigen-presenting cells. We showed that the TLR2/6 agonists were more efficient than the TLR2/1 agonists for the eradication of tumors expressing ErbB2 protein. Furthermore, mannose-targeted liposomes displayed higher therapeutic efficiency against tumor allowing treatment with decreased quantities of both TLR ligands and peptide epitopes. Our results validated that antigen-associated mannosylated liposomes combined with efficient TLR ligands are effective vectors for vaccination against tumor. In this study we developed useful tools to evaluate the vaccination efficiency of various adjuvants and/or targeting molecules and their potential synergy.

Influence of Ligand Valency on the Targeting of Immature Human Dendritic Cells by Mannosylated Liposomes

An important challenge for the development of new generations of vaccines is the efficient delivery of antigens to antigen presenting cells such as dendritic cells. In the present study we compare the interaction of plain and targeted liposomes, containing mono-, di-, and tetraantennary mannosyl lipid derivatives, with human monocyte-derived immature dendritic cells (iDCs). Whereas efficient mannose receptor-mediated endocytosis by iDCs was observed for the mannosylated liposomes, in contrast, only nonspecific interaction with little uptake was observed with plain liposomes. In accordance with the clustering effect, liposomes prepared with multibranched mannosylated lipids displayed higher binding affinity for the mannose receptor than vesicles containing the monomannosylated analogs. Importantly, we have found that dimannosylated ligands present at the surface of the liposomes were as efficient as tetramannosylated ones to engage in multidentate interactions with the mannose receptor of iDCs, resulting in both cases in an effective uptake/endocytosis. This result will greatly facilitate, from a practical standpoint, the design of mannose-targeted vaccination constructs. Moreover, we showed that mannose-mediated uptake of liposomes did not result in an activation of iDCs. Altogether, our results suggest that antigen-associated targeted liposomes containing diantennary mannosylated lipids could be effective vectors for vaccines when combined with additional DC activation signals.

Synthesis and biological activity of azasqualenes, bis-azasqualenes and derivatives

Azasqualenes, bis-azasqualenes and derivatives, designed as inhibitors of squalene 2,3-epoxide cyclase, a key enzyme in sterol biosynthesis, were synthesized and their in vitro activities against a variety of yeasts, fungi, gram-positive and gram-negative bacteria were determined. The synthesis involves a new method of squalene degradation, together with an unusual procedure for the aminative reduction of lipophilic aldehydes. A study of the structure—activity relationship was attempted for different biological parameters: anti-bacterial and anti-fungal activities (MIC), inhibition of mycelial growth (GTT), surfactant activity (CMC) and membrane perturbation activity (induction of leakage in liposomes).

Design of high energy intermediate analogues to study sterol biosynthesis in higher plants

Several enzymes of plant sterol biosynthesis involve during their catalysis postulated or demonstrated carbocationic high energy intermediates (HEI). The aim of this study was to interfere with plant sterol biosynthesis by means of rationally designed species able to mimic these carbocationic HEI. It has been demonstrated previously that the design of transition state (TS) or HEI analogues could lead to powerful and specific inhibitors of enzymes. We applied this approach to the following target enzymes: 2,3-epoxy-2,3-dihydroqualene cyclase, AdoMet-cycloartenol-C-24-methyltransferase (AdoMet CMT), cycloeucalenol-obtusifoliol isomerase (COI) and Δ8-Δ7-sterol isomerase. Very potent inhibitors have been obtained in the four cases. As an example, analogues of cycloartenol substituted at C-25 by a charged heteroatom (N, As, S) have been synthesized and shown to be able to mimic the C-25 carbocationic HEI involved in the reaction catalyzed by the AdoMet CMT. These compounds were shown to be very potent and specific inhibitors of this enzyme both in vitro (Ki=2.10−8 M, Ki/Km=10−3) and in vivo. The potent inhibitors described are powerful tools to control in vivo the sterol profile of plant cells and therefore to study the structural and functional roles of sterols in cell membranes. Moreover, these compounds constitute leader molecules of a new class of rationally designed inhibitors which could be of value in plant protection.