No abstract

In addition to its well-known role as a coenzyme in oxidation-reduction reactions, the distinct role of NAD as a precursor for molecules involved in cell regulation has been clearly established. The involvement of NAD in these regulatory processes is based on its ability to function as a donor of ADP-ribose; NAD synthesis is therefore required to avoid depletion of the intracellular pool. The rising interest in the biosynthetic routes leading to NAD formation and the highly conserved nature of the enzymes involved prompted us to reconstruct the NAD biosynthetic routes operating in distinct eukaryotic organisms. The evidence obtained from biochemical and computational analysis provides a good example of how complex metabolic pathways may evolve. In particular, it is proposed that the development of several NAD biosynthetic routes during evolution has led to partial functional redundancy, allowing a given pathway to freely acquire novel functions unrelated to NAD biosynthesis.

The conditions leading to the activation/ differentiation of T-helper (Th) cells dedicated for B-cell antibody production are still poorly characterized. We now demonstrate that interleukin-6 (IL-6) promotes the differentiation of naive T lymphocytes into helper cells able to promote B-cell activation and antibody secretion. IL-6-driven acquisition of B-cell help capacity requires expression of the signal trans- IntroductionOn activation by antigen-presenting cells (APCs), naive CD4 ϩ T-helper (Th) precursors can differentiate into functionally distinct T-cell lineages, including Th1, Th2, Th17, and regulatory T (Treg) cells. Among the critical signals that direct the induced patterns of gene expression in maturing helper T-cell subsets are cytokine-induced specific transcription factors. Interleukin-12 (IL-12) regulates Th1 differentiation through activation of the transcription factor signal transducer and activator of transcription 4 (STAT4) and T-bet, 1-3 whereas IL-4 drives Th2 differentiation through the actions of STAT6 and GATA-3. 4,5 Transforming growth factor-␤ (TGF-␤)-induced FoxP-3 is a master regulator of Treg induction, 6 and it has been recently demonstrated that development of Th17 is prompted by a combination of IL-6 plus TGF-␤ and requires expression of STAT3 and the retinoic acid-related orphan receptor ␥t (ROR␥t). 7 The help that T cells provide to B cells is a fundamental feature of mammalian immune systems that allow the production of memory B cells and long-lived plasma cells secreting high-affinity antigen-specific immunoglobulins. T-cell help to B cells was long thought to be attributable to the Th2 subset, based on the superior ability of Th2 clones to support in vitro antibody (Ab) production, and the well-documented capacity of Th2-derived cytokines (such as IL-4) to sustain B-cell growth, differentiation, and isotype switch. 8,9 However, an increasing number of experimental observations cannot be easily reconciled with this simple view. Th1 cells have been indeed shown to support B-cell responses in vitro and in vivo, [10][11][12] and mouse strains in which Th2 differentiation is strongly impaired (such as cMAF, IL-4, and STAT6 KO mice) retain the ability to secrete antibodies in response to T cell-dependent antigens. [13][14][15] More recently, T cells capable of providing help for B cells were identified in human lymphoid tissues through expression of the chemokine receptor CXCR5 and termed follicular helper T cells (T FH ) based on their anatomic localization. 16-18 Follicular CXCR5-expressing T lymphocytes appear to be particularly apt as B-cell helpers, as determined by T/B collaboration assays in vitro. These cells fail to secrete large amounts of Th1-or Th2-like cytokines, express a distinct set of genes, and can therefore not be easily classified as either Th1 or Th2. 19 It is noteworthy, however, that most T cells up-regulate CXCR5 expression on activation 20 and that not all CXCR5 ϩ cells display B-cell help capacity, 18 leaving open the question of whether Th cells for ...

*Nicotinamide phosphoribosyl transferase (Nampt)/pre-B cell colony-enhancing factor (PBEF)/visfatin is a protein displaying multiple functional properties. Originally described as a cytokine-like protein able to regulate B cell development, apoptosis, and glucose metabolism, this protein also plays an important role in NAD biosynthesis. To gain insight into its physiological role, we have generated a mouse strain expressing a conditional Nampt allele. Lack of Nampt expression strongly affects development of both T and B lymphocytes. Analysis of hemizygous cells and in vitro cell lines expressing distinct levels of Nampt illustrates the critical role of this protein in regulating intracellular NAD levels. Consequently, a clear relationship was found between intracellular Nampt levels and cell death in response to the genotoxic agent MNNG (N-methyl-N-nitro-N-nitrosoguanidine), confirming that this enzyme represents a key regulator of cell sensitivity to NAD-consuming stress secondary to poly(ADP-ribose) polymerases overactivation. By using mutant forms of this protein and a well-characterized pharmacological inhibitor (FK866), we unequivocally demonstrate that the ability of the Nampt to regulate cell viability during genotoxic stress requires its enzymatic activity. Collectively, these data demonstrate that Nampt participates in cellular resistance to genotoxic/oxidative stress, and it may confer to cells of the immune system the ability to survive during stressful situations such as inflammation.

Apolipoprotein L-I (apoL1) is a human-specific serum protein that kills Trypanosoma brucei through ionic pore formation in endosomal membranes of the parasite. The T. brucei subspecies rhodesiense and gambiense resist this lytic activity and can infect humans, causing sleeping sickness. In the case of T. b. rhodesiense, resistance to lysis involves interaction of the Serum Resistance-Associated (SRA) protein with the C-terminal helix of apoL1. We undertook a mutational and deletional analysis of the C-terminal helix of apoL1 to investigate the linkage between interaction with SRA and lytic potential for different T. brucei subspecies. We confirm that the C-terminal helix is the SRA-interacting domain. Although in E. coli this domain was dispensable for ionic pore-forming activity, its interaction with SRA resulted in inhibition of this activity. Different mutations affecting the C-terminal helix reduced the interaction of apoL1 with SRA. However, mutants in the L370-L392 leucine zipper also lost in vitro trypanolytic activity. Truncating and/or mutating the C-terminal sequence of human apoL1 like that of apoL1-like sequences of Papio anubis resulted in both loss of interaction with SRA and acquired ability to efficiently kill human serum-resistant T. b. rhodesiense parasites, in vitro as well as in transgenic mice. These findings demonstrate that SRA interaction with the C-terminal helix of apoL1 inhibits its pore-forming activity and determines resistance of T. b. rhodesiense to human serum. In addition, they provide a possible explanation for the ability of Papio serum to kill T. b. rhodesiense, and offer a perspective to generate transgenic cattle resistant to both T. b. brucei and T. b. rhodesiense.