Structural basis of SARM1 activation, substrate recognition, and inhibition by small molecules.

The NADase SARM1 (sterile alpha and TIR motif containing 1) is a key executioner of axon degeneration and a therapeutic target for several neurodegenerative conditions. We show that a potent SARM1 inhibitor undergoes base exchange with the nicotinamide moiety of nicotinamide adenine dinucleotide (NAD+) to produce the bona fide inhibitor 1AD. We report structures of SARM1 in complex with 1AD, NAD+ mimetics and the allosteric activator nicotinamide mononucleotide (NMN). NMN binding triggers reorientation of the armadillo repeat (ARM) domains, which disrupts ARM:TIR interactions and leads to formation of a two-stranded TIR domain assembly. The active site spans two molecules in these assemblies, explaining the requirement of TIR domain self-association for NADase activity and axon degeneration. Our results reveal the mechanisms of SARM1 activation and substrate binding, providing rational avenues for the design of new therapeutics targeting SARM1.

Toxin-mediated depletion of NAD and NADP drives persister formation in a human pathogen.

Toxin-antitoxin (TA) systems are widespread in bacteria and implicated in genome stability, virulence, phage defense, and persistence. TA systems have diverse activities and cellular targets, but their physiological roles and regulatory mechanisms are often unclear. Here, we show that the NatR-NatT TA system, which is part of the core genome of the human pathogen Pseudomonas aeruginosa, generates drug-tolerant persisters by specifically depleting nicotinamide dinucleotides. While actively growing P. aeruginosa cells compensate for NatT-mediated NAD+ deficiency by inducing the NAD+ salvage pathway, NAD depletion generates drug-tolerant persisters under nutrient-limited conditions. Our structural and biochemical analyses propose a model for NatT toxin activation and autoregulation and indicate that NatT activity is subject to powerful metabolic feedback control by the NAD+ precursor nicotinamide. Based on the identification of natT gain-of-function alleles in patient isolates and on the observation that NatT increases P. aeruginosa virulence, we postulate that NatT modulates pathogen fitness during infections. These findings pave the way for detailed investigations into how a toxin-antitoxin system can promote pathogen persistence by disrupting essential metabolic pathways.

SARM1 regulates NAD+-linked metabolism and select immune genes in macrophages.

Sterile alpha and HEAT/armadillo motif-containing protein (SARM1) was recently described as a NAD+-consuming enzyme and has previously been shown to regulate immune responses in macrophages. Neuronal SARM1 is known to contribute to axon degeneration due to its NADase activity. However, how SARM1 affects macrophage metabolism has not been explored. Here, we show that macrophages from Sarm1-/- mice display elevated NAD+ concentrations and lower cyclic ADP-ribose, a known product of SARM1-dependent NAD+ catabolism. Further, SARM1-deficient macrophages showed an increase in the reserve capacity of oxidative phosphorylation and glycolysis compared to WT cells. Stimulation of macrophages to a proinflammatory state by lipopolysaccharide (LPS) revealed that SARM1 restricts the ability of macrophages to upregulate glycolysis and limits the expression of the proinflammatory gene interleukin (Il) 1b, but boosts expression of anti-inflammatory Il10. In contrast, we show macrophages lacking SARM1 induced to an anti-inflammatory state by IL-4 stimulation display increased oxidative phosphorylation and glycolysis, and reduced expression of the anti-inflammatory gene, Fizz1. Overall, these data show that SARM1 fine-tunes immune gene transcription in macrophages via consumption of NAD+ and altered macrophage metabolism.

Re-equilibration of imbalanced NAD metabolism ameliorates the impact of telomere dysfunction.

Short telomeres are a principal defining feature of telomere biology disorders, such as dyskeratosis congenita (DC), for which there are no effective treatments. Here, we report that primary fibroblasts from DC patients and late generation telomerase knockout mice display lower nicotinamide adenine dinucleotide (NAD) levels, and an imbalance in the NAD metabolome that includes elevated CD38 NADase and reduced poly(ADP-ribose) polymerase and SIRT1 activities, respectively, affecting many associated biological pathways. Supplementation with the NAD precursor, nicotinamide riboside, and CD38 inhibition improved NAD homeostasis, thereby alleviating telomere damage, defective mitochondrial biosynthesis and clearance, cell growth retardation, and cellular senescence of DC fibroblasts. These findings reveal a direct, underlying role of NAD dysregulation when telomeres are short and underscore its relevance to the pathophysiology and interventions of human telomere-driven diseases.

Structure of the Streptococcus pyogenes NAD+ Glycohydrolase Translocation Domain and Its Essential Role in Toxin Binding to Oropharyngeal Keratinocytes.

The emergence and continued dominance of a Streptococcus pyogenes (group A Streptococcus, GAS) M1T1 clonal group is temporally correlated with acquisition of genomic sequences that confer high level expression of cotoxins streptolysin O (SLO) and NAD+-glycohydrolase (NADase). Experimental infection models have provided evidence that both toxins are important contributors to GAS virulence. SLO is a cholesterol-dependent pore-forming toxin capable of lysing virtually all types of mammalian cells. NADase, which is composed of an N-terminal translocation domain and C-terminal glycohydrolase domain, acts as an intracellular toxin that depletes host cell energy stores. NADase is dependent on SLO for internalization into epithelial cells, but its mechanism of interaction with the cell surface and details of its translocation mechanism remain unclear. In this study we found that NADase can bind oropharyngeal epithelial cells independently of SLO. This interaction is mediated by both domains of the toxin. We determined by NMR the structure of the translocation domain to be a ß-sandwich with a disordered N-terminal region. The folded region of the domain has structural homology to carbohydrate binding modules. We show that excess NADase inhibits SLO-mediated hemolysis and binding to epithelial cells in vitro, suggesting NADase and SLO have shared surface receptors. This effect is abrogated by disruption of a putative carbohydrate binding site on the NADase translocation domain. Our data are consistent with a model whereby interactions of the NADase glycohydrolase domain and translocation domain with SLO and the cell surface increase avidity of NADase binding and facilitate toxin-toxin and toxin-cell surface interactions. IMPORTANCE NADase and streptolysin O (SLO) are secreted toxins important for pathogenesis of group A Streptococcus, the agent of strep throat and severe invasive infections. The two toxins interact in solution and mutually enhance cytotoxic activity. We now find that NADase is capable of binding to the surface of human cells independently of SLO. Structural analysis of the previously uncharacterized translocation domain of NADase suggests that it contains a carbohydrate binding module. The NADase translocation domain and SLO appear to recognize similar glycan structures on the cell surface, which may be one mechanism through which NADase enhances SLO pore-forming activity during infection. Our findings provide new insight into the NADase toxin and its functional interactions with SLO during streptococcal infection.

A new biochemistry connecting pathogen detection to induced defense in plants.

Plant cell surface and intracellular immune receptors recognizing pathogen attack utilize the same defense machineries to mobilize resistance. New genetic, protein structural and biochemical information on receptor activation and signaling is transforming understanding of how their shared defense network operates. We discuss the biochemical activities of two classes of intracellular nucleotide-binding/leucine-rich repeat (NLR) receptor - one forming a Ca2+ channel, the other an NADase enzyme - which define engagement of enhanced disease susceptibility 1 (EDS1)-family heterodimers and cofunctioning helper NLRs (RNLs) to connect receptor systems and amplify defenses. Toll-interleukin-1 receptor (TIR) domain NLR receptors and TIR-domain proteins, with a capacity to produce NAD+-derived small molecules, require EDS1 dimers and RNLs for defense induction. The TIR-driven EDS1/RNL modules emerge as central elements in Ca2+ -based immunity signaling initiated by receptors outside and inside host cells.

SARM1 Ablation Is Protective and Preserves Spatial Vision in an In Vivo Mouse Model of Retinal Ganglion Cell Degeneration.

The challenge of developing gene therapies for genetic forms of blindness is heightened by the heterogeneity of these conditions. However, mechanistic commonalities indicate key pathways that may be targeted in a gene-independent approach. Mitochondrial dysfunction and axon degeneration are common features of many neurodegenerative conditions including retinal degenerations. Here we explore the neuroprotective effect afforded by the absence of sterile alpha and Toll/interleukin-1 receptor motif-containing 1 (SARM1), a prodegenerative NADase, in a rotenone-induced mouse model of retinal ganglion cell loss and visual dysfunction. Sarm1 knockout mice retain visual function after rotenone insult, displaying preservation of photopic negative response following rotenone treatment in addition to significantly higher optokinetic response measurements than wild type mice following rotenone. Protection of spatial vision is sustained over time in both sexes and is accompanied by increased RGC survival and additionally preservation of axonal density in optic nerves of Sarm1-/- mice insulted with rotenone. Primary fibroblasts extracted from Sarm1-/- mice demonstrate an increased oxygen consumption rate relative to those from wild type mice, with significantly higher basal, maximal and spare respiratory capacity. Collectively, our data indicate that Sarm1 ablation increases mitochondrial bioenergetics and confers histological and functional protection in vivo in the mouse retina against mitochondrial dysfunction, a hallmark of many neurodegenerative conditions including a variety of ocular disorders.

How activated NLRs induce anti-microbial defenses in plants.

Plants utilize cell-surface localized and intracellular leucine-rich repeat (LRR) immune receptors to detect pathogens and to activate defense responses, including transcriptional reprogramming and the initiation of a form of programmed cell death of infected cells. Cell death initiation is mainly associated with the activation of nucleotide-binding LRR receptors (NLRs). NLRs recognize the presence or cellular activity of pathogen-derived virulence proteins, so-called effectors. Effector-dependent NLR activation leads to the formation of higher order oligomeric complexes, termed resistosomes. Resistosomes can either form potential calcium-permeable cation channels at cellular membranes and initiate calcium influxes resulting in activation of immunity and cell death or function as NADases whose activity is needed for the activation of downstream immune signaling components, depending on the N-terminal domain of the NLR protein. In this mini-review, the current knowledge on the mechanisms of NLR-mediated cell death and resistance pathways during plant immunity is discussed.

Diverse NADase effector families mediate interbacterial antagonism via the type VI secretion system.

The bacterial type VI secretion system (T6SS) mediates antagonistic cell-cell interactions between competing Gram-negative bacteria. In plant-beneficial bacteria, this pathway has been shown to suppress the growth of bacterial pathogens; however, the identification and mode of action of T6SS effector proteins that mediate this protective effect remain poorly defined. Here, we identify two previously uncharacterized effectors required for interbacterial antagonism by the plant commensal bacterium Pseudomonas protegens Consistent with the established effector-immunity paradigm for antibacterial T6SS substrates, the toxic activities of these effectors are neutralized by adjacently encoded cognate immunity determinants. Although one of these effectors, RhsA, belongs to the family of DNase enzymes, the activity of the other was not apparent from its sequence. To determine the mechanism of toxicity of this latter effector, we determined its 1.3 Å crystal structure in complex with its immunity protein and found that it resembles NAD(P)+-degrading enzymes. In line with this structural similarity, biochemical characterization of this effector, termed Tne2 (Type VI secretion NADase effector family 2), demonstrates that it possesses potent NAD(P)+ hydrolase activity. Tne2 is the founding member of a widespread family of interbacterial NADases predicted to transit not only the Gram-negative T6SS but also the Gram-positive type VII secretion system, a pathway recently implicated in interbacterial competition among Firmicutes. Together, this work identifies new T6SS effectors employed by a plant commensal bacterium to antagonize its competitors and broadly implicates NAD(P)+-hydrolyzing enzymes as substrates of interbacterial conflict pathways found in diverse bacterial phyla.

Effect of Phosphatase Activity of the Control of Virulence Sensor (CovS) on Clindamycin-Mediated Streptolysin O Production in Group A Streptococcus.

Severe manifestations of group A Streptococcus (GAS) infections are associated with massive tissue destruction and high mortality. Clindamycin (CLI), a bacterial protein synthesis inhibitor, is recommended for treating patients with severe invasive GAS infection. Nonetheless, the subinhibitory concentration of CLI induces the production of GAS virulent exoproteins, such as streptolysin O (SLO) and NADase, which would enhance bacterial virulence and invasiveness. A better understanding of the molecular mechanism of how CLI triggers GAS virulence factor expression will be critical to develop appropriate therapeutic approaches. The present study shows that CLI activates SLO and NADase expressions in the emm1-type CLI-susceptible wild-type strain but not in covS or control of virulence sensor (CovS) phosphatase-inactivated mutants. Supplementation with Mg2+, which is a CovS phosphatase inhibitor, inhibits the CLI-mediated SLO upregulation in a dose-dependent manner in CLI-susceptible and CLI-resistant strains. These results not only reveal that the phosphorylation of response regulator CovR is essential for responding to CLI stimuli, but also suggest that inhibiting the phosphatase activity of CovS could be a potential strategy for the treatment of invasive GAS infection with CLI.

Acidic stress enhances CovR/S-dependent gene repression through activation of the covR/S promoter in emm1-type group A Streptococcus.

Streptococcus pyogenes (group A Streptococcus) is a clinically important gram-positive bacterium that causes severe diseases with high mortality. Spontaneous mutations in genes encoding the CovR/CovS two-component regulatory system have been shown to derepress expression of virulence factors and are significantly associated with invasiveness of infections. Sensor kinase CovS senses environmental signals and then regulates the levels of phosphorylated CovR. In addition, CovS is responsible for survival of group A Streptococcus under acidic stress. How this system regulates the expression of CovR-controlled genes under acidic stress is not clear. This study shows that the expression of CovR-controlled genes, including hasA, ska, and slo, is repressed under acidic conditions by a CovS-dependent mechanism. Inactivation of CovS kinase activity or CovR protein phosphorylation derepresses the transcription of these genes under acidic conditions, suggesting that the phosphorylation of CovR is required for the repression of the CovR-controlled genes. Furthermore, the promoter activity of the covR/covS operon (pcov) was upregulated after 15min of incubation under acidic conditions. Replacement of pcov with a constitutively activated promoter abrogated the acidic-stress-dependent repression of the genes, indicating that the pH-dependent pcov activity is directly involved in the repression of CovR-controlled genes. In summary, the present study shows that inactivation of CovS not only derepresses CovR-controlled genes but also abrogates the acidic-stress-dependent repression of the genes; these phenomena may significantly increase bacterial virulence during infection.

Association of CovRS Two-Component Regulatory System with NADase Induction by Clindamycin Treatment in Streptococcus pyogenes.

Administration of high-dose clindamycin (CLI) and penicillin is recommended for the treatment of streptococcal toxic shock syndrome (STSS). However, CLI-resistant strains have been identified worldwide. In this study, some CLI-resistant strains demonstrated increased extracellular activity of the NAD-glycohydrolase (NADase) exotoxin following CLI treatment. These results support our previous conclusion that CLI-susceptible and CLI-resistant Streptococcus pyogenes strains exhibit CLI-dependent NADase induction. Furthermore, we investigated the mechanism of this phenomenon using 13 types of two-component sensor knockout strains derived from the CLI-susceptible strain 1529 that has a CLI-dependent NADase induction phenotype. Among the knockout strains, only 1529ΔcovS lost the phenotype. Additionally, 1529ΔspeB, 1529Δmga, and 1529Δrgg retained the CLI-dependent NADase induction phenotype. These findings indicate that CovS is related to this phenotype in a SpeB-independent manner.

Potential Therapeutic Interventions Targeting NAD+ Metabolism for ALS.

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease affecting both upper and lower motor neurons. While there have been many potential factors implicated for ALS development, such as oxidative stress and mitochondrial dysfunction, no exact mechanism has been determined at this time. Nicotinamide adenine dinucleotide (NAD+) is one of the most abundant metabolites in mammalian cells and is crucial for a broad range of cellular functions from DNA repair to energy homeostasis. NAD+ can be synthesized from three different intracellular pathways, but it is the NAD+ salvage pathway that generates the largest proportion of NAD+. Impaired NAD+ homeostasis has been connected to aging and neurodegenerative disease-related dysfunctions. In ALS mice, NAD+ homeostasis is potentially disrupted prior to the appearance of physical symptoms and is significantly reduced in the nervous system at the end stage. Treatments targeting NAD+ metabolism, either by administering NAD+ precursor metabolites or small molecules that alter NAD+-dependent enzyme activity, have shown strong beneficial effects in ALS disease models. Here, we review the therapeutic interventions targeting NAD+ metabolism for ALS and their effects on the most prominent pathological aspects of ALS in animal and cell models.

Structural insights into autoinhibition and activation of defense-associated sirtuin protein.

Bacterial defense-associated sirtuin 2 (DSR2) proteins harbor an N-terminal sirtuin (SIR2) domain degrading NAD+. DSR2 from Bacillus subtilis 29R is autoinhibited and unable to hydrolyze NAD+ in the absence of phage infection. A tail tube protein (TTP) of phage SPR activates the DSR2 while a DSR2-inhibiting protein of phage SPbeta, known as DSAD1 (DSR anti-defense 1), inactivates the DSR2. Although DSR2 structures in complexed with TTP and DSAD1, respectively, have been reported recently, the autoinhibition and activation mechanisms remain incompletely understood. Here, we present cryo-electron microscopy structures of the DSR2-NAD+ complex in autoinhibited state and the in vitro assembled DSR2-TFD (TTP tube-forming domain) complex in activated state. The DSR2-NAD+ complex reveals that the autoinhibited DSR2 assembles into an inactive tetramer, binding NAD+ through a distinct pocket situated outside active site. Binding of TFD into cavities within the sensor domains of DSR2 triggers a conformational change in SIR2 regions, activating its NADase activity, whereas the TTP ß-sandwich domain (BSD) is flexible and does not contribute to the activation process. The activated form of DSR2 exists as tetramers and dimers, with the tetramers exhibiting more NADase activity. Overall, our results extend the current understanding of autoinhibition and activation of DSR2 immune proteins.

Ecto-CD38-NADase inhibition modulates cardiac metabolism and protects mice against doxorubicin-induced cardiotoxicity.

AIMS: Doxorubicin (DXR) is a chemotherapeutic agent that causes dose-dependent cardiotoxicity. Recently, it has been proposed that the NADase CD38 may play a role in doxorubicin-induced cardiotoxicity (DIC). CD38 is the main NAD+-catabolizing enzyme in mammalian tissues. Interestingly, in the heart, CD38 is mostly expressed as an ecto-enzyme that can be targeted by specific inhibitory antibodies. The goal of the present study is to characterize the role of CD38 ecto-enzymatic activity in cardiac metabolism and the development of DIC. METHODS AND RESULTS: Using both a transgenic animal model and a non-cytotoxic enzymatic anti-CD38 antibody, we investigated the role of CD38 and its ecto-NADase activity in DIC in pre-clinical models. First, we observed that DIC was prevented in the CD38 catalytically inactive (CD38-CI) transgenic mice. Both left ventricular systolic function and exercise capacity were decreased in wild-type but not in CD38-CI mice treated with DXR. Second, blocking CD38-NADase activity with the specific antibody 68 (Ab68) likewise protected mice against DIC and decreased DXR-related mortality by 50%. A reduction of DXR-induced mitochondrial dysfunction, energy deficiency, and inflammation gene expression were identified as the main mechanisms mediating the protective effects. CONCLUSION: NAD+-preserving strategies by inactivation of CD38 via a genetic or a pharmacological-based approach improve cardiac energetics and reduce cardiac inflammation and dysfunction otherwise seen in an acute DXR cardiotoxicity model.

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.

Nucleic acid-induced NADase activation of a short Sir2-associated prokaryotic Argonaute system.

Inhibition of nucleic acid targets is mediated by Argonaute (Ago) proteins guided by RNA or DNA. Although the mechanisms underpinning the functions of eukaryotic and "long" prokaryotic Ago proteins (pAgos) are well understood, those for short pAgos remain enigmatic. Here, we determine two cryoelectron microscopy structures of short pAgos in association with the NADase-domain-containing protein Sir2-APAZ from Geobacter sulfurreducens (GsSir2/Ago): the guide RNA-target DNA-loaded GsSir2/Ago quaternary complex (2.58 Å) and the dimer of the quaternary complex (2.93Å). These structures show that the nucleic acid binding causes profound conformational changes that result in disorder or partial dissociation of the Sir2 domain, suggesting that it adopts a NADase-active conformation. Subsequently, two RNA-/DNA-loaded GsSir2/Ago complexes form a dimer through their MID domains, further enhancing NADase activity through synergistic effects. The findings provide a structural basis for short-pAgo-mediated defense against invading nucleic acids.

NAD+ Consuming Enzymes: Involvement in Therapies and Prevention of Human Diseases.

Neuroprotection is one of the hot topics in medicine. Alzheimer's disease, amyotrophic lateral sclerosis, retinal pigment epithelial (RPE) degeneration, and axonal degeneration have been studied for the involvement of NAD depletion. Localized NAD+ depletion could lead to overactivation and crowding of local NAD+ salvage pathways. It has been stated that NAD+ depletion caused by PARPs and PAR cycling has been related to metabolic diseases and cancer. Additionally, it is now acknowledged that SARM1 dependent NAD+ depletion causes axon degeneration. New targeted therapeutics, such as SARM1 inhibitors, and NAD+ salvage drugs will help alleviate the dysfunctions affecting cell life and death in neurodegeneration as well as in metabolic diseases and cancer.

The curious case of SARM1: Dr. Jekyll and Mr. Hyde in cell death and immunity?

Sterile alpha and toll/interleukin-1 receptor motif-containing protein 1 (SARM1) was first identified as a novel ortholog of Drosophila protein CG7915 and was subsequently placed as the fifth member of the human TIR-containing adaptor protein. SARM1 holds a unique position in this family where, unlike other members, it downregulates NFκB activity in response to immunogenic stimulation, interacts with another member of the family, TRIF, to negatively regulate its function, and it also mediates cell death responses. Over the past decade, SARM1 has emerged as one of the primary mediators of programmed axonal degeneration and this robust regulation of axonal degeneration-especially in models of peripheral neuropathy and traumatic injury-makes it an attractive target for therapeutic intervention. The TIR domain of SARM1 possesses an intrinsic NADase activity resulting in cellular energy deficits within the axons, a striking deviation from its other family members of human TLR adaptors. Interestingly, the TIR NADase activity, as seen in SARM1, is also observed in several prokaryotic TIR-containing proteins where they are involved in immune evasion once within the host. Although the immune function of SARM1 is yet to be conclusively discerned, this closeness in function with the prokaryotic TIR-domain containing proteins, places it at an interesting juncture of evolution raising questions about its origin and function in cell death and immunity. In this review, we discuss how a conserved immune adaptor protein like SARM1 switches to a pro-neurodegenerative function and the evolutionarily significance of the process.

Assembly and Architecture of NLR Resistosomes and Inflammasomes.

Nucleotide-binding and leucine-rich repeat (NLR) proteins are critical intracellular immune receptors in both animals and plants. Perception of pathogen-derived or stress-associated signals induces NLR oligomerization to form multiprotein complexes called inflammasomes in animals or resistosomes in plants to mediate host immune response. Significant progress has been made during the past few years in our understanding of NLR biology, particularly the structural perspective of these two types of NLR-containing complexes. In this article, we review the latest advances in our structural knowledge of how NLR inflammasomes and resistosomes are activated and assembled and how the structural information provides insight into their distinct mechanisms of action. Commonalities and differences between NLR inflammasomes and resistosomes are also discussed.