Evolution of fungal tuberculosis necrotizing toxin (TNT) domain-containing enzymes reveals divergent adaptations to enhance NAD cleavage.

Tuberculosis necrotizing toxin (TNT) is a protein domain discovered on the outer membrane of Mycobacterium tuberculosis (Mtb), and the fungal pathogen Aspergillus fumigatus. TNT domains have pure NAD(P) hydrolytic activity, setting them apart from other NAD-cleaving domains such as ADP-ribosyl cyclase and Toll/interleukin-1 receptor homology (TIR) domains which form a wider set of products. Importantly, the Mtb TNT domain has been shown to be involved in immune evasion via depletion of the intracellular NAD pool of macrophages. Therefore, an intriguing hypothesis is that TNT domains act as "NAD killers" in host cells facilitating pathogenesis. Here, we explore the phylogenetic distribution of TNT domains and detect their presence solely in bacteria and fungi. Within fungi, we discerned six TNT clades. In addition, X-ray crystallography and AlphaFold2 modeling unveiled clade-specific strategies to promote homodimer stabilization of the fungal enzymes, namely, Ca2+ binding, disulfide bonds, or hydrogen bonds. We show that dimer stabilization is a requirement for NADase activity and that the group-specific strategies affect the active site conformation, thereby modulating enzyme activity. Together, these findings reveal the evolutionary lineage of fungal TNT enzymes, corroborating the hypothesis of them being pure extracellular NAD (eNAD) cleavers, with possible involvement in microbial warfare and host immune evasion.

Damage-Associated Molecular Patterns and Systemic Signaling.

Cellular damage inflicted by wounding, pathogen infection, and herbivory releases a variety of host-derived metabolites, degraded structural components, and peptides into the extracellular space that act as alarm signals when perceived by adjacent cells. These so-called damage-associated molecular patterns (DAMPs) function through plasma membrane localized pattern recognition receptors to regulate wound and immune responses. In plants, DAMPs act as elicitors themselves, often inducing immune outputs such as calcium influx, reactive oxygen species generation, defense gene expression, and phytohormone signaling. Consequently, DAMP perception results in a priming effect that enhances resistance against subsequent pathogen infections. Alongside their established function in local tissues, recent evidence supports a critical role of DAMP signaling in generation and/or amplification of mobile signals that induce systemic immune priming. Here, we summarize the identity, signaling, and synergy of proposed and established plant DAMPs, with a focus on those with published roles in systemic signaling.

Differences in Extracellular NAD+ and NMN Metabolism on the Surface of Vascular Endothelial Cells.

The disruption of the metabolism of extracellular NAD+ and NMN may affect related signaling cascades and pathologies, such as cardiovascular or respiratory system diseases. We aimed to study NAD+ and NMN hydrolysis on surface endothelial cells of diverse origins and with genetically modified nucleotide catabolism pathways. We tested lung endothelial cells isolated from C57BL/6 J wild-type (WT) and C57BL/6 J CD73 knockout (CD73 KO) mice, the transfected porcine iliac artery endothelial cell line (PIEC) with the human E5NT gene for CD73 (PIEC CD73), and a mock-transfected control (PIEC MOCK), as well as HMEC-1 and H5V cells. Substrate conversion into the product was followed by high-performance liquid chromatography (HPLC). We showed profound differences in extracellular NAD+ and NMN metabolism related to the vessel origin, species diversity, and type of culture. We also confirmed the involvement of CD38 and CD73 in NAD+ and NMN cleavage.

A Methodological Approach Using rAAV Vectors Encoding Nanobody-Based Biologics to Evaluate ARTC2.2 and P2X7 In Vivo.

On murine T cells, mono-ADP ribosyltransferase ARTC2.2 catalyzes ADP-ribosylation of various surface proteins when nicotinamide adenine dinucleotide (NAD+) is released into the extracellular compartment. Covalent ADP-ribosylation of the P2X7 receptor by ARTC2.2 thereby represents an additional mechanism of activation, complementary to its triggering by extracellular ATP. P2X7 is a multifaceted receptor that may represents a potential target in inflammatory, and neurodegenerative diseases, as well as in cancer. We present herein an experimental approach using intramuscular injection of recombinant AAV vectors (rAAV) encoding nanobody-based biologics targeting ARTC2.2 or P2X7. We demonstrate the ability of these in vivo generated biologics to potently and durably block P2X7 or ARTC2.2 activities in vivo, or in contrast, to potentiate NAD+- or ATP-induced activation of P2X7. We additionally demonstrate the ability of rAAV-encoded functional heavy chain antibodies to elicit long-term depletion of T cells expressing high levels of ARTC2.2 or P2X7. Our approach of using rAAV to generate functional nanobody-based biologics in vivo appears promising to evaluate the role of ARTC2.2 and P2X7 in murine acute as well as chronic disease models.

Degradation of Extracellular NAD+ Intermediates in Cultures of Human HEK293 Cells.

Nicotinamide adenine dinucleotide (NAD) is an essential redox carrier, whereas its degradation is a key element of important signaling pathways. Human cells replenish their NAD contents through NAD biosynthesis from extracellular precursors. These precursors encompass bases nicotinamide (Nam) and nicotinic acid and their corresponding nucleosides nicotinamide riboside (NR) and nicotinic acid riboside (NAR), now collectively referred to as vitamin B3. In addition, extracellular NAD+ and nicotinamide mononucleotide (NMN), and potentially their deamidated counterparts, nicotinic acid adenine dinucleotide (NAAD) and nicotinic acid mononucleotide (NAMN), may serve as precursors of intracellular NAD. However, it is still debated whether nucleotides enter cells directly or whether they are converted to nucleosides and bases prior to uptake into cells. Here, we studied the metabolism of extracellular NAD+ and its derivatives in human HEK293 cells using normal and serum-free culture medium. Using medium containing 10% fetal bovine serum (FBS), mono- and dinucleotides were degraded to the corresponding nucleosides. In turn, the nucleosides were cleaved to their corresponding bases. Degradation was also observed in culture medium alone, in the absence of cells, indicating that FBS contains enzymatic activities which degrade NAD+ intermediates. Surprisingly, NR was also rather efficiently hydrolyzed to Nam in the absence of FBS. When cultivated in serum-free medium, HEK293 cells efficiently cleaved NAD+ and NAAD to NMN and NAMN. NMN exhibited rather high stability in cell culture, but was partially metabolized to NR. Using pharmacological inhibitors of plasma membrane transporters, we also showed that extracellular cleavage of NAD+ and NMN to NR is a prerequisite for using these nucleotides to maintain intracellular NAD contents. We also present evidence that, besides spontaneous hydrolysis, NR is intensively metabolized in cell culture by intracellular conversion to Nam. Our results demonstrate that both the cultured cells and the culture medium mediate a rather active conversion of NAD+ intermediates. Consequently, in studies of precursor supplementation and uptake, the culture conditions need to be carefully defined.

Exogenous Nicotinamide Adenine Dinucleotide Induces Resistance to Citrus Canker in Citrus.

Nicotinamide adenine dinucleotide (NAD) is a universal electron carrier that participates in important intracellular metabolic reactions and signaling events. Interestingly, emerging evidence in animals indicates that cellular NAD can be actively or passively released into the extracellular space, where it is processed or perceived by ectoenzymes or cell-surface receptors. We have recently shown in Arabidopsis thaliana that exogenous NAD induces defense responses, that pathogen infection leads to release of NAD into the extracellular space at concentrations sufficient for defense activation, and that depletion of extracellular NAD (eNAD) by transgenic expression of the human NAD-hydrolyzing ectoenzyme CD38 inhibits plant immunity. We therefore hypothesize that, during plant-microbe interactions, NAD is released from dead or dying cells into the extracellular space where it interacts with adjacent naïve cells' surface receptors, which in turn activate downstream immune signaling. However, it is currently unknown whether eNAD signaling is unique to Arabidopsis or the Brassicaceae family. In this study, we treated citrus plants with exogenous NAD+ and tested NAD+-induced transcriptional changes and disease resistance. Our results show that NAD+ induces profound transcriptome changes and strong resistance to citrus canker, a serious citrus disease caused by the bacterial pathogen Xanthomonas citri subsp. citri (Xcc). Furthermore, NAD+-induced resistance persists in new flushes emerging after removal of the tissues previously treated with NAD+. Finally, NAD+ treatment primes citrus tissues, resulting in a faster and stronger induction of multiple salicylic acid pathway genes upon subsequent Xcc infection. Taken together, these results indicate that exogenous NAD+ is able to induce immune responses in citrus and suggest that eNAD may also be an elicitor in this woody plant species.

Extracellular pyridine nucleotides as immune elicitors in arabidopsis.

The pyridine nucleotides nicotinamide adenine dinucleotide (NAD) and NAD phosphate (NADP) are coenzymes that function in both metabolic reactions and intracellular signaling. Emerging evidence from animal research indicates that NAD(P) also acts in the extracellular space (ECS). We have shown in the model plant Arabidopsis that (1) exogenous NAD(P) induces immune responses, (2) pathogen infection causes leakage of intracellular NAD(P) into the extracellular fluid at concentrations sufficient to induce immune responses, and (3) removal of extracellular NAD(P) [eNAD(P)] by expressing the human NAD(P)-metabolizing ectoenzyme CD38 partially compromises systemic acquired resistance. Based on these results, we hypothesize that eNAD(P) is a novel damage-associated molecular pattern (DAMP) in plants; during plant-microbe interaction, intracellular NAD(P) is released from dead or dying cells into the ECS where it interacts with the adjacent healthy cells' surface receptors/targets, which in turn activate downstream specific immune signaling pathways. Our recent identification of LecRK-I.8, a lectin receptor kinase, as the first cell surface NAD+-binding receptor has provided compelling evidence for this hypothesis. Further identification of cell surface eNAD(P) receptors/targets and their downstream signaling components in Arabidopsis as well as determination of the generality of eNAD(P) signaling in crops will help establish eNAD(P) as a conserved DAMP in plants.

A lectin receptor kinase as a potential sensor for extracellular nicotinamide adenine dinucleotide in Arabidopsis thaliana.

Nicotinamide adenine dinucleotide (NAD+) participates in intracellular and extracellular signaling events unrelated to metabolism. In animals, purinergic receptors are required for extracellular NAD+ (eNAD+) to evoke biological responses, indicating that eNAD+ may be sensed by cell-surface receptors. However, the identity of eNAD+-binding receptors still remains elusive. Here, we identify a lectin receptor kinase (LecRK), LecRK-I.8, as a potential eNAD+ receptor in Arabidopsis. The extracellular lectin domain of LecRK-I.8 binds NAD+ with a dissociation constant of 436.5 ± 104.8 nM, although much higher concentrations are needed to trigger in vivo responses. Mutations in LecRK-I.8 inhibit NAD+-induced immune responses, whereas overexpression of LecRK-I.8 enhances the Arabidopsis response to NAD+. Furthermore, LecRK-I.8 is required for basal resistance against bacterial pathogens, substantiating a role for eNAD+ in plant immunity. Our results demonstrate that lectin receptors can potentially function as eNAD+-binding receptors and provide direct evidence for eNAD+ being an endogenous signaling molecule in plants.

The human NAD metabolome: Functions, metabolism and compartmentalization.

The metabolism of NAD has emerged as a key regulator of cellular and organismal homeostasis. Being a major component of both bioenergetic and signaling pathways, the molecule is ideally suited to regulate metabolism and major cellular events. In humans, NAD is synthesized from vitamin B3 precursors, most prominently from nicotinamide, which is the degradation product of all NAD-dependent signaling reactions. The scope of NAD-mediated regulatory processes is wide including enzyme regulation, control of gene expression and health span, DNA repair, cell cycle regulation and calcium signaling. In these processes, nicotinamide is cleaved from NAD(+) and the remaining ADP-ribosyl moiety used to modify proteins (deacetylation by sirtuins or ADP-ribosylation) or to generate calcium-mobilizing agents such as cyclic ADP-ribose. This review will also emphasize the role of the intermediates in the NAD metabolome, their intra- and extra-cellular conversions and potential contributions to subcellular compartmentalization of NAD pools.

Scant Extracellular NAD Cleaving Activity of Human Neutrophils is Down-Regulated by fMLP via FPRL1.

Extracellular nicotinamide adenine dinucleotide (NAD) cleaving activity of a particular cell type determines the rate of the degradation of extracellular NAD with formation of metabolites in the vicinity of the plasma membrane, which has important physiological consequences. It is yet to be elucidated whether intact human neutrophils have any extracellular NAD cleaving activity. In this study, with a simple fluorometric assay utilizing 1,N(6)-ethenoadenine dinucleotide (etheno-NAD) as the substrate, we have shown that intact peripheral human neutrophils have scant extracellular etheno-NAD cleaving activity, which is much less than that of mouse bone marrow neutrophils, mouse peripheral neutrophils, human monocytes and lymphocytes. With high performance liquid chromatography (HPLC), we have identified that ADP-ribose (ADPR) is the major extracellular metabolite of NAD degradation by intact human neutrophils. The scant extracellular etheno-NAD cleaving activity is decreased further by N-formyl-methionine-leucine-phenylalanine (fMLP), a chemoattractant for neutrophils. The fMLP-mediated decrease in the extracellular etheno-NAD cleaving activity is reversed by WRW4, a potent FPRL1 antagonist. These findings show that a much less extracellular etheno-NAD cleaving activity of intact human neutrophils compared to other immune cell types is down-regulated by fMLP via a low affinity fMLP receptor FPRL1.