Browsing by keyword "NAD"
Now showing items 1-4 of 4
-
A Lymphocyte Surface Protein Produces the Signaling Molecule Poly (ADP-ribose) from NADElucidation of signaling pathways that prevent immune cells from damaging self-tissue could help target diseases like lupus and juvenile diabetes. Through one such pathway, NAD and its metabolites appear to inhibit T cells of the immune system. NAD is a substrate for two enzyme families that covalently transfer ADP-ribose from NAD to acceptor proteins -- mono-ADP-ribosyl transferases (mARTs) and poly(ADP-ribose) polymerases (PARPs). PARPs distinguish themselves by polymerizing ADP-ribose on the acceptor proteins. Despite differences in amino acid sequences, mARTs and PARPs have similar structural elements in their catalytic cores. Here we report that in the presence of NAD, ART2, a mART and T cell surface protein, forms ADP-ribose polymers on an arginine in a crucial loop of its catalytic core. ART2 appears to be the first hybrid between the mARTs and PARPs, and structural data suggest a mechanism for polymerization activity. The data suggest that signaling with NAD metabolites like ADP-ribose may be a more versatile process than previously recognized, and that molecules like ART2 may have the potential to participate in novel immune cell signaling pathways.
-
Crystal structures of homoserine dehydrogenase suggest a novel catalytic mechanism for oxidoreductasesThe structure of the antifungal drug target homoserine dehydrogenase (HSD) was determined from Saccharomyces cerevisiae in apo and holo forms, and as a ternary complex with bound products, by X-ray diffraction. The three forms show that the enzyme is a dimer, with each monomer composed of three regions, the nucleotide-binding region, the dimerization region and the catalytic region. The dimerization and catalytic regions have novel folds, whereas the fold of the nucleotide-binding region is a variation on the Rossmann fold. The novel folds impose a novel composition and arrangement of active site residues when compared to all other currently known oxidoreductases. This observation, in conjunction with site-directed mutagenesis of active site residues and steady-state kinetic measurements, suggest that HSD exhibits a new variation on dehydrogenase chemistry.
-
Identification of the first noncompetitive SARM1 inhibitorsSterile Alpha and Toll Interleukin Receptor Motif-containing protein 1 (SARM1) is a key therapeutic target for diseases that exhibit Wallerian-like degeneration; Wallerian degeneration is characterized by degeneration of the axon distal to the site of injury. These diseases include traumatic brain injury, peripheral neuropathy, and neurodegenerative diseases. SARM1 promotes neurodegeneration by catalyzing the hydrolysis of NAD(+) to form a mixture of ADPR and cADPR. Notably, SARM1 knockdown prevents degeneration, indicating that SARM1 inhibitors will likely be efficacious in treating these diseases. Consistent with this hypothesis is the observation that NAD(+) supplementation is axoprotective. To identify compounds that block the NAD(+) hydrolase activity of SARM1, we developed and performed a high-throughput screen (HTS). This HTS assay exploits an NAD(+) analog, etheno-NAD(+) (ENAD) that fluoresces upon cleavage of the nicotinamide moiety. From this screen, we identified berberine chloride and zinc chloride as the first noncompetitive inhibitors of SARM1. Though modest in potency, the noncompetitive mode of inhibition, suggests the presence of an allosteric binding pocket on SARM1 that can be targeted for future therapeutic development. Additionally, zinc inhibition and site-directed mutagenesis reveals that cysteines 629 and 635 are critical for SARM1 catalysis, highlighting these sites for the design of inhibitors targeting SARM1.
-
Insights into the Role of SARM1 in Pathological Neuron DeathTraumatic brain injury, peripheral neuropathies, and other neurodegenerative diseases exhibit diverse clinical manifestations but are connected by their underlying trigger, axonal degeneration. These diseases cause extensive morbidity and mortality worldwide, as treatments are palliative and no curative treatments exist. SARM1 has recently emerged as a therapeutic target for these diseases as knockdown prevents axonal degeneration and ameliorates disease prognosis. Later, it was shown that SARM1 hydrolyzes NAD+ in response to degenerative stressors. Given that NAD+ supplementation delays axonal degeneration, we expect therapeutically targeting SARM1 will be efficacious for neurodegenerative diseases. However, the design of SARM1 therapeutics is limited by the dearth of knowledge surrounding its NAD+ hydrolase activity and active structural state. Illuminating this black box has been hindered by technical difficulties in obtaining pure active protein. To circumvent these issues, I began by studying SARM1 in lysates. I synthesized truncated constructs and developed three different assays, which enabled me to characterize the kinetic activity. I also established a high–throughput screening pipeline to identify inhibitors and screened >4,000 compounds. Recently, I identified additives (i.e., PEG and citrate) that activate SARM1 by ~2,000–fold, making it feasible to study the purified protein. I found that the additives enhance activity by inducing SARM1 to form a multimeric precipitate. To further interrogate the role multimerization plays in activity, I performed detailed mutagenesis and cell culture studies. The insights from this thesis have aided in our understanding of this elusive enzyme and provided strategic direction for future SARM1 investigation and drug development.
