SAMHD1 Phosphorylation at T592 Regulates Cellular Localization and S-phase Progression.

SAMHD1 activity is regulated by a network of mechanisms including phosphorylation, oxidation, oligomerization, and others. Significant questions remain about the effects of phosphorylation on SAMHD1 function and activity. We investigated the effects of a SAMHD1 T592E phosphorylation mimic on its cellular localization, catalytic activity, and cell cycle progression. We found that the SAMHD1 T592E is a catalytically active enzyme that is inhibited by protein oxidation. SAMHD1 T592E is retained in the nucleus at higher levels than the wild-type protein during growth factor-mediated signaling. This nuclear localization protects SAMHD1 from oxidation by cytoplasmic reactive oxygen species. The SAMHD1 T592E phosphomimetic further inhibits the cell cycle S/G2 transition. This has significant implications for SAMHD1 function in regulating innate immunity, antiviral response and DNA replication.

Identification of Inhibitors of the dNTP Triphosphohydrolase SAMHD1 Using a Novel and Direct High-Throughput Assay.

The dNTP triphosphohydrolase SAMHD1 is a regulator of cellular dNTP pools. Given its central role in nucleotide metabolism, SAMHD1 performs important functions in cellular homeostasis, cell cycle regulation, and innate immunity. It therefore represents a high-profile target for small molecule drug design. SAMHD1 has a complex mechanism of catalytic activation that makes the design of an activating compound challenging. However, an inhibitor of SAMHD1 could serve multiple therapeutic roles, including the potentiation of antiviral and anticancer drug regimens. The lack of high-throughput screens that directly measure SAMHD1 catalytic activity has impeded efforts to identify inhibitors of SAMHD1. Here we describe a novel high-throughput screen that directly measures SAMHD1 catalytic activity. This assay results in a colorimetric end point that can be read spectrophotometrically and utilizes bis(4-nitrophenyl) phosphate as the substrate and Mn2+ as the activating cation that facilitates catalysis. When used to screen a library of Food and Drug Administration-approved drugs, this HTS identified multiple novel compounds that inhibited SAMHD1 dNTPase activity at micromolar concentrations.

SAMHD1: Recurring roles in cell cycle, viral restriction, cancer, and innate immunity.

Sterile alpha motif and histidine-aspartic acid domain-containing protein 1 (SAMHD1) is a deoxynucleotide triphosphate (dNTP) hydrolase that plays an important role in the homeostatic balance of cellular dNTPs. Its emerging role as an effector of innate immunity is affirmed by mutations in the SAMHD1 gene that cause the severe autoimmune disease, Aicardi-Goutieres syndrome (AGS) and that are linked to cancer. Additionally, SAMHD1 functions as a restriction factor for retroviruses, such as HIV. Here, we review the current biochemical and biological properties of the enzyme including its structure, activity, and regulation by post-translational modifications in the context of its cellular function. We outline open questions regarding the biology of SAMHD1 whose answers will be important for understanding its function as a regulator of cell cycle progression, genomic integrity, and in autoimmunity.

The SAMHD1 dNTP Triphosphohydrolase Is Controlled by a Redox Switch.

AIMS: Proliferative signaling involves reversible posttranslational oxidation of proteins. However, relatively few molecular targets of these modifications have been identified. We investigate the role of protein oxidation in regulation of SAMHD1 catalysis. RESULTS: Here we report that SAMHD1 is a major target for redox regulation of nucleotide metabolism and cell cycle control. SAMHD1 is a triphosphate hydrolase, whose function involves regulation of deoxynucleotide triphosphate pools. We demonstrate that the redox state of SAMHD1 regulates its catalytic activity. We have identified three cysteine residues that constitute an intrachain disulfide bond "redox switch" that reversibly inhibits protein tetramerization and catalysis. We show that proliferative signals lead to SAMHD1 oxidation in cells and oxidized SAMHD1 is localized outside of the nucleus. Innovation and Conclusions: SAMHD1 catalytic activity is reversibly regulated by protein oxidation. These data identify a previously unknown mechanism for regulation of nucleotide metabolism by SAMHD1. Antioxid. Redox Signal. 27, 1317-1331.