NADase Activity Research Articles

A duplex structure of SARM1 octamers stabilized by a new inhibitor.

In recent years, there has been growing interest in SARM1 as a potential breakthrough drug target for treating various pathologies of axon degeneration. SARM1-mediated axon degeneration relies on its TIR domain NADase activity, but recent structural data suggest that the non-catalytic ARM domain could also serve as a pharmacological site as it has an allosteric inhibitory function. Here, we screened for synthetic small molecules that inhibit SARM1, and tested a selected set of these compounds in a DRG axon degeneration assay. Using cryo-EM, we found that one of the newly discovered inhibitors, a calmidazolium designated TK106, not only stabilizes the previously reported inhibited conformation of the octamer, but also a meta-stable structure: a duplex of octamers (16 protomers), which we have now determined to 4.0Å resolution. In the duplex, each ARM domain protomer is engaged in lateral interactions with neighboring protomers, and is further stabilized by contralateral contacts with the opposing octamer ring. Mutagenesis of the duplex contact sites leads to a moderate increase in SARM1 activation in cultured cells. Based on our data we propose that the duplex assembly constitutes an additional auto-inhibition mechanism that tightly prevents pre-mature activation and axon degeneration.

General Phenotype of NADase Induction by CLI Treatment in Streptococcus pyogenes.

The administration of high-dose clindamycin (CLI) along with penicillin is recommended for the treatment of streptococcal toxic-shock syndrome (STSS). However, we previously reported that a "subinhibitory dose" of CLI induced the expression of the NAD-glycohydrolase (NADase) exotoxin in an emm1-type Streptococcus pyogenes 1529 strain isolated from an STSS patient. In this study, we examine NADase induction by CLI treatment using an extracellular NADase activity assay instead of the previous two-dimensional gel electrophoresis assay. The examination revealed that CLI administration can induce NADase expression in a dose-dependent manner. We analyzed 23 CLI-susceptible strains (5 emm1 strains, 6 emm3 strains, 3 emm4 strains, 1 emm6 strain, 3 emm12 strains, 1 emm28 strain, and 4 emm89 strains), and 19 of the 23 strains showed similar NADase induction phenotypes to that shown in strain 1529. These results indicate that NADase induction by CLI treatment is not restricted to specific strains and it could be a standard phenotype among CLI-susceptible S. pyogenes strains. We also analyzed four CLI-resistant strains. All four strains showed increased extracellular NADase activities at high concentrations of CLI that did not inhibit bacterial growth. These results indicated that the subinhibitory dose of CLI was not the critical factor for NADase induction.

Selective inhibitors of SARM1 targeting an allosteric cysteine in the autoregulatory ARM domain

The nicotinamide adenine dinucleotide hydrolase (NADase) sterile alpha toll/interleukin receptor motif containing-1 (SARM1) acts as a central executioner of programmed axon death and is a possible therapeutic target for neurodegenerative disorders. While orthosteric inhibitors of SARM1 have been described, this multidomain enzyme is also subject to intricate forms of autoregulation, suggesting the potential for allosteric modes of inhibition. Previous studies have identified multiple cysteine residues that support SARM1 activation and catalysis, but which of these cysteines, if any, might be selectively targetable by electrophilic small molecules remains unknown. Here, we describe the chemical proteomic discovery of a series of tryptoline acrylamides that site-specifically and stereoselectively modify cysteine-311 (C311) in the noncatalytic, autoregulatory armadillo repeat (ARM) domain of SARM1. These covalent compounds inhibit the NADase activity of WT-SARM1, but not C311A or C311S SARM1 mutants, show a high degree of proteome-wide selectivity for SARM1_C311 and stereoselectively block vincristine- and vacor-induced neurite degeneration in primary rodent dorsal root ganglion neurons. Our findings describe selective, covalent inhibitors of SARM1 targeting an allosteric cysteine, pointing to a potentially attractive therapeutic strategy for axon degeneration-dependent forms of neurological disease.

Natural variants of human SARM1 cause both intrinsic and dominant loss-of-function influencing axon survival

SARM1 is a central executioner of programmed axon death, and this role requires intrinsic NAD(P)ase or related enzyme activity. A complete absence of SARM1 robustly blocks axon degeneration in mice, but even a partial depletion confers meaningful protection. Since axon loss contributes substantially to the onset and progression of multiple neurodegenerative disorders, lower inherent SARM1 activity is expected to reduce disease susceptibility in some situations. We, therefore, investigated whether there are naturally occurring SARM1 alleles within the human population that encode SARM1 variants with loss-of-function. Out of the 18 natural SARM1 coding variants we selected as candidates, we found that 10 display loss-of-function in three complimentary assays: they fail to robustly deplete NAD in transfected HEK 293T cells; they lack constitutive and NMN-induced NADase activity; and they fail to promote axon degeneration in primary neuronal cultures. Two of these variants are also able to block axon degeneration in primary culture neurons in the presence of endogenous, wild-type SARM1, indicative of dominant loss-of-function. These results demonstrate that SARM1 loss-of-function variants occur naturally in the human population, and we propose that carriers of these alleles will have different degrees of reduced susceptibility to various neurological conditions.

TIR-catalyzed ADP-ribosylation reactions produce signaling molecules for plant immunity.

Plant pathogen-activated immune signaling by nucleotide-binding leucine-rich repeat (NLR) receptors with an N-terminal Toll/interleukin-1 receptor (TIR) domain converges on Enhanced Disease Susceptibility 1 (EDS1) and its direct partners, Phytoalexin Deficient 4 (PAD4) or Senescence-Associated Gene 101 (SAG101). TIR-encoded nicotinamide adenine dinucleotide hydrolase (NADase) produces signaling molecules to promote exclusive EDS1-PAD4 and EDS1-SAG101 interactions with helper NLR subclasses. In this work, we show that TIR-containing proteins catalyze adenosine diphosphate (ADP)-ribosylation of adenosine triphosphate (ATP) and ADP ribose (ADPR) through ADPR polymerase-like and NADase activity, forming ADP-ribosylated ATP (ADPr-ATP) and ADPr-ADPR (di-ADPR), respectively. Specific binding of ADPr-ATP or di-ADPR allosterically promotes EDS1-SAG101 interaction with helper NLR N requirement gene 1A (NRG1A) in vitro and in planta. Our data reveal an enzymatic activity of TIRs that enables specific activation of the EDS1-SAG101-NRG1 immunity branch.

Identification and receptor mechanism of TIR-catalyzed small molecules in plant immunity.

Plant nucleotide-binding leucine-rich repeat-containing (NLR) receptors with an N-terminal Toll/interleukin-1 receptor (TIR) domain sense pathogen effectors to enable TIR-encoded nicotinamide adenine dinucleotide hydrolase (NADase) activity for immune signaling. TIR-NLR signaling requires the helper NLRs N requirement gene 1 (NRG1), Activated Disease Resistance 1 (ADR1), and Enhanced Disease Susceptibility 1 (EDS1), which forms a heterodimer with each of its paralogs Phytoalexin Deficient 4 (PAD4) and Senescence-Associated Gene 101 (SAG101). Here, we show that TIR-containing proteins catalyze the production of 2'-(5''-phosphoribosyl)-5'-adenosine monophosphate (pRib-AMP) and diphosphate (pRib-ADP) in vitro and in planta. Biochemical and structural data demonstrate that EDS1-PAD4 is a receptor complex for pRib-AMP and pRib-ADP, which allosterically promote EDS1-PAD4 interaction with ADR1-L1 but not NRG1A. Our study identifies TIR-catalyzed pRib-AMP and pRib-ADP as a missing link in TIR signaling through EDS1-PAD4 and as likely second messengers for plant immunity.

Distinct developmental and degenerative functions of SARM1 require NAD+ hydrolase activity.

SARM1 is the founding member of the TIR-domain family of NAD+ hydrolases and the central executioner of pathological axon degeneration. SARM1-dependent degeneration requires NAD+ hydrolysis. Prior to the discovery that SARM1 is an enzyme, SARM1 was studied as a TIR-domain adaptor protein with non-degenerative signaling roles in innate immunity and invertebrate neurodevelopment, including at the Drosophila neuromuscular junction (NMJ). Here we explore whether the NADase activity of SARM1 also contributes to developmental signaling. We developed transgenic Drosophila lines that express SARM1 variants with normal, deficient, and enhanced NADase activity and tested their function in NMJ development. We find that NMJ overgrowth scales with the amount of NADase activity, suggesting an instructive role for NAD+ hydrolysis in this developmental signaling pathway. While degenerative and developmental SARM1 signaling share a requirement for NAD+ hydrolysis, we demonstrate that these signals use distinct upstream and downstream mechanisms. These results identify SARM1-dependent NAD+ hydrolysis as a heretofore unappreciated component of developmental signaling. SARM1 now joins sirtuins and Parps as enzymes that regulate signal transduction pathways via mechanisms that involve NAD+ cleavage, greatly expanding the potential scope of SARM1 TIR NADase functions.

TIR domains of plant immune receptors are 2′,3′-cAMP/cGMP synthetases mediating cell death

2',3'-cAMP is a positional isomer of the well-established second messenger 3',5'-cAMP, but little is known about the biology of this noncanonical cyclic nucleotide monophosphate (cNMP). Toll/interleukin-1 receptor (TIR) domains of nucleotide-binding leucine-rich repeat (NLR) immune receptors have the NADase function necessary but insufficient to activate plant immune responses. Here, we show that plant TIR proteins, besides being NADases, act as 2',3'-cAMP/cGMP synthetases by hydrolyzing RNA/DNA. Structural data show that a TIR domain adopts distinct oligomers with mutually exclusive NADase and synthetase activity. Mutations specifically disrupting the synthetase activity abrogate TIR-mediated cell death in Nicotiana benthamiana (Nb), supporting an important role for these cNMPs in TIR signaling. Furthermore, the Arabidopsis negative regulator of TIR-NLR signaling, NUDT7, displays 2',3'-cAMP/cGMP but not 3',5'-cAMP/cGMP phosphodiesterase activity and suppresses cell death activity of TIRs in Nb. Our study identifies a family of 2',3'-cAMP/cGMP synthetases and establishes a critical role for them in plant immune responses.

Lack of the immune adaptor molecule SARM1 accelerates disease in prion infected mice and is associated with increased mitochondrial respiration and decreased expression of NRF2.

Prion diseases are a group of fatal, transmissible neurodegenerative diseases of mammals. In the brain, axonal loss and neuronal death are prominent in prion infection, but the mechanisms remain poorly understood. Sterile alpha and heat/Armadillo motif 1 (SARM1) is a protein expressed in neurons of the brain that plays a critical role in axonal degeneration. Following damage to axons, it acquires an NADase activity that helps to regulate mitochondrial health by breaking down NAD+, a molecule critical for mitochondrial respiration. SARM1 has been proposed to have a protective effect in prion disease, and we hypothesized that it its role in regulating mitochondrial energetics may be involved. We therefore analyzed mitochondrial respiration in SARM1 knockout mice (SARM1KO) and wild-type mice inoculated either with prions or normal brain homogenate. Pathologically, disease was similar in both strains of mice, suggesting that SARM1 mediated axonal degradation is not the sole mechanism of axonal loss during prion disease. However, mitochondrial respiration was significantly increased and disease incubation time accelerated in prion infected SARM1KO mice when compared to wild-type mice. Increased levels of mitochondrial complexes II and IV and decreased levels of NRF2, a potent regulator of reactive oxygen species, were also apparent in the brains of SARM1KO mice when compared to wild-type mice. Our data suggest that SARM1 slows prion disease progression, likely by regulating mitochondrial respiration, which may help to mitigate oxidative stress via NRF2.

NADase activity found in bacterial TIR proteins may aid in innate immune evasion

Abstract Pathogenic microbes have evolved various mechanisms to evade recognition by host immune responses, which can lead to the prevalence of disease and infection. Many strains of pathogenic bacteria contain genes encoding Toll/Interleukin-1 receptor (TIR) domain-containing proteins that function through the inhibition of TLR and myeloid differentiation factor 88 (MyD88) signaling. Previous research has also revealed that prokaryotic TIR domains may regulate bacterial and host metabolic pathways by acting as NAD+ glycohydrolases (NADases). We hypothesize that this NADase activity may contribute to the role of bacterial TIRs as virulence factors. In order to test this hypothesis, we observed the effects of TIR NADase activity on the binding interactions between bacterial TIRs and host MyD88, and on the ability of bacterial TIRs to block NF-kB signaling. For this, we have identified amino acids predicted to play a role in TIR NADase activity and have expressed and purified recombinant bacterial TIR proteins that have incorporated these mutations. We then tested the NADase activity of these mutants in comparison with that of wild-type TIR proteins and TIRs with a mutation known to inhibit NADase activity. We are now using TIRs with deficiencies in NADase activity in pull-down assays to test their binding to MyD88 and utilizing RT-PCR to analyze the effects of these mutant TIRs on innate cell activation. This information will allow us to gain a deeper understanding into the relationship between NADase activity in bacterial TIRs and their virulence activity and can potentially uncover new mechanisms for pathogenic evasion of host immune defenses. Supported by grant from NIH (R25 GM119970 04)

SARM1 Depletion Slows Axon Degeneration in a CNS Model of Neurotropic Viral Infection.

Zika virus (ZIKV) is a neurotropic flavivirus recently linked to congenital ZIKV syndrome in children and encephalitis and Guillain-Barré syndrome in adults. Neurotropic viruses often use axons to traffic to neuronal or glial cell somas where they either remain latent or replicate and proceed to infect new cells. Consequently, it has been suggested that axon degeneration could represent an evolutionarily conserved mechanism to limit viral spread. Whilst it is not known if ZIKV transits in axons, we previously reported that ZIKV infection of glial cells in a murine spinal cord-derived cell culture model of the CNS is associated with a profound loss of neuronal cell processes. This, despite that postmitotic neurons are relatively refractory to infection and death. Here, we tested the hypothesis that ZIKV-associated degeneration of neuronal processes is dependent on activation of Sterile alpha and armadillo motif-containing protein 1 (SARM1), an NADase that acts as a central executioner in a conserved axon degeneration pathway. To test this, we infected wild type and Sarm1 homozygous or heterozygous null cell cultures with ZIKV and examined NAD+ levels as well as the survival of neurons and their processes. Unexpectedly, ZIKV infection led to a rapid SARM1-independent reduction in NAD+. Nonetheless, the subsequent profound loss of neuronal cell processes was SARM1-dependent and was preceded by early changes in the appearance of β-tubulin III staining. Together, these data identify a role for SARM1 in the pathogenesis of ZIKV infection, which may reflect SARM1's conserved prodegenerative function, independent of its NADase activity.

Products of gut microbial Toll/interleukin-1 receptor domain NADase activities in gnotobiotic mice and Bangladeshi children with malnutrition.

SummaryPerturbed gut microbiome development has been linked to childhood malnutrition. Here, we characterize bacterial Toll/interleukin-1 receptor (TIR) protein domains that metabolize nicotinamide adenine dinucleotide (NAD), a co-enzyme with far-reaching effects on human physiology. A consortium of 26 human gut bacterial strains, representing the diversity of TIRs observed in the microbiome and the NAD hydrolase (NADase) activities of a subset of 152 bacterial TIRs assayed in vitro, was introduced into germ-free mice. Integrating mass spectrometry and microbial RNA sequencing (RNA-seq) with consortium membership manipulation disclosed that a variant of cyclic-ADPR (v-cADPR-x) is a specific product of TIR NADase activity and a prominent, colonization-discriminatory, taxon-specific metabolite. Guided by bioinformatic analyses of biochemically validated TIRs, we find that acute malnutrition is associated with decreased fecal levels of genes encoding TIRs known or predicted to generate v-cADPR-x, as well as decreased levels of the metabolite itself. These results underscore the need to consider microbiome TIR NADases when evaluating NAD metabolism in the human holobiont.

Life-or-death decisions in plant immunity.

Upon pathogen challenge, plant cells can mount defense not only by triggering programmed cell death (PCD) to limit pathogen growth, but also by secreting immune signals to activate subsequent organism-scale defense responses. Recent advances in the study of plant immune mechanisms have found that pathogen-induced oligomerization of immune receptors is a common 'on' switch for the normally self-inhibitory proteins. The resulting 'resistosome' triggers PCD through the formation of a calcium channel or a NADase. Synergy between different receptor-mediated signaling pathways appears to be required for sustained immune induction to trigger PCD of infected cells. In the neighboring cells, PCD is inhibited through the production of immune signal salicylic acid (SA) which mediates degradation of PCD-inducing immune components in biomolecular condensates. Future work is required to connect the resistosome-mediated channel formation and the NADase activity to the downstream regulation of immune execution.

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.

The CD38 glycohydrolase and the NAD sink: implications for pathological conditions.

Nicotinamide adenine dinucleotide (NAD) acts as a cofactor in several oxidation-reduction (redox) reactions and is a substrate for a number of nonredox enzymes. NAD is fundamental to a variety of cellular processes including energy metabolism, cell signaling, and epigenetics. NAD homeostasis appears to be of paramount importance to health span and longevity, and its dysregulation is associated with multiple diseases. NAD metabolism is dynamic and maintained by synthesis and degradation. The enzyme CD38, one of the main NAD-consuming enzymes, is a key component of NAD homeostasis. The majority of CD38 is localized in the plasma membrane with its catalytic domain facing the extracellular environment, likely for the purpose of controlling systemic levels of NAD. Several cell types express CD38, but its expression predominates on endothelial cells and immune cells capable of infiltrating organs and tissues. Here we review potential roles of CD38 in health and disease and postulate ways in which CD38 dysregulation causes changes in NAD homeostasis and contributes to the pathophysiology of multiple conditions. Indeed, in animal models the development of infectious diseases, autoimmune disorders, fibrosis, metabolic diseases, and age-associated diseases including cancer, heart disease, and neurodegeneration are associated with altered CD38 enzymatic activity. Many of these conditions are modified in CD38-deficient mice or by blocking CD38 NADase activity. In diseases in which CD38 appears to play a role, CD38-dependent NAD decline is often a common denominator of pathophysiology. Thus, understanding dysregulation of NAD homeostasis by CD38 may open new avenues for the treatment of human diseases.

Crystal structure of the Toll/interleukin-1 receptor (TIR) domain of IL-1R10 provides structural insights into TIR domain signalling.

The Toll/interleukin-1 receptor (TIR) domains are key innate immune signalling modules. Here, we present the crystal structure of the TIR domain of human interleukin-1 receptor 10 (IL-1R10), also called interleukin 1 receptor accessory protein like 2. It is similar to that of IL-1R9 (IL-1RAPL1) but shows significant structural differences to those from Toll-like receptors (TLRs) and the adaptor proteins MyD88 adaptor-like protein (MAL) and MyD88. Interactions of TIR domains in their respective crystals and the higher-order assemblies (MAL and MyD88) reveal the presence of a common 'BCD surface', suggesting its functional significance. We also show that the TIR domains of IL-1R10 and IL-1R9 lack NADase activity, consistent with their structures. Our study provides a foundation for unravelling the functions of IL-1R9 and IL-1R10.

Constitutively active SARM1 variants that induce neuropathy are enriched in ALS patients

BackgroundIn response to injury, neurons activate a program of organized axon self-destruction initiated by the NAD+ hydrolase, SARM1. In healthy neurons SARM1 is autoinhibited, but single amino acid changes can abolish autoinhibition leading to constitutively active SARM1 enzymes that promote degeneration when expressed in cultured neurons.MethodsTo investigate whether naturally occurring human variants might disrupt SARM1 autoinhibition and potentially contribute to risk for neurodegenerative disease, we assayed the enzymatic activity of all 42 rare SARM1 alleles identified among 8507 amyotrophic lateral sclerosis (ALS) patients and 9671 controls. We then intrathecally injected mice with virus expressing SARM1 constructs to test the capacity of an ALS-associated constitutively active SARM1 variant to promote neurodegeneration in vivo.ResultsTwelve out of 42 SARM1 missense variants or small in-frame deletions assayed exhibit constitutive NADase activity, including more than half of those that are unique to the ALS patients or that occur in multiple patients. There is a > 5-fold enrichment of constitutively active variants among patients compared to controls. Expression of constitutively active ALS-associated SARM1 alleles in cultured dorsal root ganglion (DRG) neurons is pro-degenerative and cytotoxic. Intrathecal injection of an AAV expressing the common SARM1 reference allele is innocuous to mice, but a construct harboring SARM1V184G, the constitutively active variant found most frequently among the ALS patients, causes axon loss, motor dysfunction, and sustained neuroinflammation.ConclusionsThese results implicate rare hypermorphic SARM1 alleles as candidate genetic risk factors for ALS and other neurodegenerative conditions.

SARM1 participates in axonal degeneration and mitochondrial dysfunction in prion disease.

Prion disease represents a group of fatal neurogenerative diseases in humans and animals that are associated with energy loss, axonal degeneration, and mitochondrial dysfunction. Axonal degeneration is an early hallmark of neurodegeneration and is triggered by SARM1. We found that depletion or dysfunctional mutation of SARM1 protected against NAD+ loss, axonal degeneration, and mitochondrial functional disorder induced by the neurotoxic peptide PrP106-126. NAD+ supplementation rescued prion-triggered axonal degeneration and mitochondrial dysfunction and SARM1 overexpression suppressed this protective effect. NAD+ supplementation in PrP106-126-incubated N2a cells, SARM1 depletion, and SARM1 dysfunctional mutation each blocked neuronal apoptosis and increased cell survival. Our results indicate that the axonal degeneration and mitochondrial dysfunction triggered by PrP106-126 are partially dependent on SARM1 NADase activity. This pathway has potential as a therapeutic target in the early stages of prion disease.

Enrichment of SARM1 alleles encoding variants with constitutively hyperactive NADase in patients with ALS and other motor nerve disorders.

SARM1, a protein with critical NADase activity, is a central executioner in a conserved programme of axon degeneration. We report seven rare missense or in-frame microdeletion human SARM1 variant alleles in patients with amyotrophic lateral sclerosis (ALS) or other motor nerve disorders that alter the SARM1 auto-inhibitory ARM domain and constitutively hyperactivate SARM1 NADase activity. The constitutive NADase activity of these seven variants is similar to that of SARM1 lacking the entire ARM domain and greatly exceeds the activity of wild-type SARM1, even in the presence of nicotinamide mononucleotide (NMN), its physiological activator. This rise in constitutive activity alone is enough to promote neuronal degeneration in response to otherwise non-harmful, mild stress. Importantly, these strong gain-of-function alleles are completely patient-specific in the cohorts studied and show a highly significant association with disease at the single gene level. These findings of disease-associated coding variants that alter SARM1 function build on previously reported genome-wide significant association with ALS for a neighbouring, more common SARM1 intragenic single nucleotide polymorphism (SNP) to support a contributory role of SARM1 in these disorders. A broad phenotypic heterogeneity and variable age-of-onset of disease among patients with these alleles also raises intriguing questions about the pathogenic mechanism of hyperactive SARM1 variants.

Live imaging reveals the cellular events downstream of SARM1 activation.

SARM1 is an inducible NAD+ hydrolase that triggers axon loss and neuronal cell death in the injured and diseased nervous system. While SARM1 activation and enzyme function are well defined, the cellular events downstream of SARM1 activity but prior to axonal demise are much less well understood. Defects in calcium, mitochondria, ATP, and membrane homeostasis occur in injured axons, but the relationships among these events have been difficult to disentangle because prior studies analyzed large collections of axons in which cellular events occur asynchronously. Here, we used live imaging of mouse sensory neurons with single axon resolution to investigate the cellular events downstream of SARM1 activity. Our studies support a model in which SARM1 NADase activity leads to an ordered sequence of events from loss of cellular ATP, to defects in mitochondrial movement and depolarization, followed by calcium influx, externalization of phosphatidylserine, and loss of membrane permeability prior to catastrophic axonal self-destruction.