SARM1, an Enzyme Involved in Axon Degeneration, Catalyzes Multiple Activities through a Ternary Complex Mechanism.

Sterile alpha and toll/interleukin receptor (TIR) motif containing protein 1 (SARM1) is an NAD+ hydrolase and cyclase involved in axonal degeneration. In addition to NAD+ hydrolysis and cyclization, SARM1 catalyzes a base exchange reaction between nicotinic acid (NA) and NADP+ to generate NAADP, which is a potent calcium signaling molecule. Herein, we describe efforts to characterize the hydrolysis, cyclization, and base exchange activities of TIR-1, the Caenorhabditis elegans ortholog of SARM1; TIR-1 also catalyzes NAD(P)+ hydrolysis and/or cyclization and regulates axonal degeneration in worms. We show that the catalytic domain of TIR-1 undergoes a liquid-to-solid phase transition that regulates not only the hydrolysis and cyclization reactions but also the base exchange reaction. We define the substrate specificities of the reactions, demonstrate that cyclization and base exchange reactions occur within the same pH range, and establish that TIR-1 uses a ternary complex mechanism. Overall, our findings will aid drug discovery efforts and provide insight into the mechanism of recently described inhibitors.

The chemical biology of NAD+ regulation in axon degeneration.

During axon degeneration, NAD+ levels are largely controlled by two enzymes: nicotinamide mononucleotide adenylyltransferase 2 (NMNAT2) and sterile alpha and toll interleukin motif containing protein 1 (SARM1). NMNAT2, which catalyzes the formation of NAD+ from NMN and ATP, is actively degraded leading to decreased NAD+ levels. SARM1 activity further decreases the concentration of NAD+ by catalyzing its hydrolysis to form nicotinamide and a mixture of ADPR and cADPR. Notably, SARM1 knockout mice show decreased neurodegeneration in animal models of axon degeneration, highlighting the therapeutic potential of targeting this novel NAD+ hydrolase. This review discusses recent advances in the SARM1 field, including SARM1 structure, regulation, and catalysis as well as the identification of the first SARM1 inhibitors.

Pathogen infection and cholesterol deficiency activate the C. elegans p38 immune pathway through a TIR-1/SARM1 phase transition.

Intracellular signaling regulators can be concentrated into membrane-free, higher ordered protein assemblies to initiate protective responses during stress - a process known as phase transition. Here, we show that a phase transition of the Caenorhabditis elegans Toll/interleukin-1 receptor domain protein (TIR-1), an NAD+ glycohydrolase homologous to mammalian sterile alpha and TIR motif-containing 1 (SARM1), underlies p38 PMK-1 immune pathway activation in C. elegans intestinal epithelial cells. Through visualization of fluorescently labeled TIR-1/SARM1 protein, we demonstrate that physiologic stresses, both pathogen and non-pathogen, induce multimerization of TIR-1/SARM1 into visible puncta within intestinal epithelial cells. In vitro enzyme kinetic analyses revealed that, like mammalian SARM1, the NAD+ glycohydrolase activity of C. elegans TIR-1 is dramatically potentiated by protein oligomerization and a phase transition. Accordingly, C. elegans with genetic mutations that specifically block either multimerization or the NAD+ glycohydrolase activity of TIR-1/SARM1 fail to induce p38 PMK phosphorylation, are unable to increase immune effector expression, and are dramatically susceptible to bacterial infection. Finally, we demonstrate that a loss-of-function mutation in nhr-8, which alters cholesterol metabolism and is used to study conditions of sterol deficiency, causes TIR-1/SARM1 to oligomerize into puncta in intestinal epithelial cells. Cholesterol scarcity increases p38 PMK-1 phosphorylation, primes immune effector induction in a manner that requires TIR-1/SARM1 oligomerization and its intrinsic NAD+ glycohydrolase activity, and reduces pathogen accumulation in the intestine during a subsequent infection. These data reveal a new adaptive response that allows a metazoan host to anticipate pathogen threats during cholesterol deprivation, a time of relative susceptibility to infection. Thus, a phase transition of TIR-1/SARM1 as a prerequisite for its NAD+ glycohydrolase activity is strongly conserved across millions of years of evolution and is essential for diverse physiological processes in multiple cell types.

From worms to humans, animals have developed various strategies ­ including immune defences ­ to shield themselves from disease-causing microbes. A type of roundworm, called C. elegans, lives in environments rich in microbes, so it needs effective immune defences to protect itself. The roundworms share a key regulatory pathway with mammals that helps to control their immune responses. This so-called p38 pathway relies on proteins that interact with each other to activate protective immune defences. Proteins contain different regions or domains that can give them a certain function. For example, proteins with a region called TIR play important roles in immune defences in both animals and plants. One such protein, called SARM1, is unique among animal and plant proteins in that it is an enzyme, which cleaves an important metabolite in the cell. In C. elegans, the SARM1 homolog, TIR-1, controls the p38 pathway during infection, but how TIR-1 activates it is unclear. To find out more, Peterson, Icso et al. modified C. elegans to generate a fluorescent form of TIR-1 and infected the worms with bacteria. Imaging techniques revealed that infection caused TIR-1 in gut cells to cluster into organized structures, which increases the enzymatic activity of the protein to activate the p38 immune pathway. Moreover, stress situations, such as cholesterol nutrient withdrawal, activated the p38 pathway in the same way. This adaptive stress response allows the animal to defend itself against pathogen threats during times, when they are most susceptible to infections. Cells in the gut provide a primary line of defence against infectious bacteria and are important for maintaining a healthy gut immune system. When the mechanisms for pathogen sensing and immune maintenance are disrupted, it can lead to inflammation and higher risk of infection. Peterson, Icso et al. show how a key regulator of gut immunity, TIR-1, provides protection in C. elegans, which may suggest that SARM1 could have a similar role in mammals.

A phase transition enhances the catalytic activity of SARM1, an NAD+ glycohydrolase involved in neurodegeneration.

Sterile alpha and toll/interleukin receptor (TIR) motif-containing protein 1 (SARM1) is a neuronally expressed NAD+ glycohydrolase whose activity is increased in response to stress. NAD+ depletion triggers axonal degeneration, which is a characteristic feature of neurological diseases. Notably, loss of SARM1 is protective in murine models of peripheral neuropathy and traumatic brain injury. Herein, we report that citrate induces a phase transition that enhances SARM1 activity by ~2000-fold. This phase transition can be disrupted by mutating a residue involved in multimerization, G601P. This mutation also disrupts puncta formation in cells. We further show that citrate induces axonal degeneration in C. elegans that is dependent on the C. elegans orthologue of SARM1 (TIR-1). Notably, citrate induces the formation of larger puncta indicating that TIR-1/SARM1 multimerization is essential for degeneration in vivo. These findings provide critical insights into SARM1 biology with important implications for the discovery of novel SARM1-targeted therapeutics.

Initial Kinetic Characterization of Sterile Alpha and Toll/Interleukin Receptor Motif-Containing Protein 1.

Sterile alpha and toll/interleukin receptor (TIR) motif-containing protein 1 (SARM1) plays a pivotal role in triggering the neurodegenerative processes that underlie peripheral neuropathies, traumatic brain injury, and neurodegenerative diseases. Importantly, SARM1 knockdown or knockout prevents degeneration, thereby demonstrating that SARM1 is a promising therapeutic target. Recently, SARM1 was shown to promote neurodegeneration via its ability to hydrolyze NAD+, forming nicotinamide and ADP ribose (ADPR). Herein, we describe the initial kinetic characterization of full-length SARM1, as well as the truncated constructs corresponding to the SAM1-2TIR and TIR domains, highlighting the distinct challenges that have complicated efforts to characterize this enzyme. Moreover, we show that bacterially expressed full-length SARM1 (kcat/KM = 6000 ± 2000 M-1 s-1) is at least as active as the TIR domain alone (kcat/KM = 1500 ± 300 M-1 s-1). Finally, we show that the SARM1 hydrolyzes NAD+ via an ordered uni-bi reaction in which nicotinamide is released prior to ADPR.