The HIV-1 restriction factor sterile α-motif/histidine-aspartate domain-containing protein 1 (SAMHD1) is a tetrameric protein that catalyzes the hydrolysis of all dNTPs to the deoxynucleoside and tripolyphosphate, which effectively depletes the dNTP substrates of HIV reverse transcriptase. Here, we establish that SAMHD1 is activated by GTP binding to guanine-specific activator sites (A1) as well as coactivation by substrate dNTP binding to a distinct set of nonspecific activator sites (A2). Combined activation by GTP and dNTPs results in a long-lived tetrameric form of SAMHD1 that persists for hours, even after activating nucleotides are withdrawn from the solution. These results reveal an ordered model for assembly of SAMHD1 tetramer from its inactive monomer and dimer forms, where GTP binding to the A1 sites generates dimer and dNTP binding to the A2 and catalytic sites generates active tetramer. Thus, cellular regulation of active SAMHD1 is not determined by GTP alone but instead, the levels of all dNTPs and the generation of a persistent tetramer that is not in equilibrium with free activators. The significance of the long-lived activated state is that SAMHD1 can remain active long after dNTP pools have been reduced to a level that would lead to inactivation. This property would be important in resting CD4 + T cells, where dNTP pools are reduced to nanomolar levels to restrict infection by HIV-1. dNTP induced oligomerization | enzyme catalysis | innate immunity T he steady-state composition and concentration of deoxynucleotide triphosphate pools in mammalian cells are highly regulated because of the mutagenic consequences of dNTP imbalances in dividing cells (1, 2) as well as the important antiviral effects of dNTP pool depletion in quiescent cells (3,4). In all cell types, the ultimate pool balance is determined by dNTP-dependent regulatory pathways that affect the activities of enzymes involved in both synthesis and degradation of dNTPs (5-7). The most important highly up-regulated synthetic enzyme during S phase of dividing cells is the R1/R2 isoform of ribonucleotide triphosphate reductase, which ensures that dNTP precursors are plentiful for DNA synthesis (8). However, in quiescent cells of the immune system (resting CD4 + T cells, macrophages, and dendritic cells), where dNTP pools are ∼10-fold lower than dividing cells, the ultimate pool levels are likely determined by a balance between the activities of the R1/p53R2 isoform of ribonucleotide triphosphate reductase and the degradative dNTP triphosphohydrolase sterile α-motif/histidineaspartate domain-containing protein 1 (SAMHD1) (9). The highly dynamic nature of dNTP pools demands finely tuned mechanisms for feedback regulation of these enzymes by dNTPs as well as coarse regulatory mechanisms (posttranslational modifications, transcriptional regulation, and proteasomal targeting) that serve to turn these activities on and off at appropriate stages of the cell cycle and in specific cell types (10, 11). dNTP triphosphohydrolase enzymes, such as SAMHD1,...

The HIV-1 restriction factor SAMHD1 is a tetrameric enzyme activated by guanine nucleotides with dNTP triphosphate hydrolase activity (dNTPase). In addition to this established activity, there have been a series of conflicting reports as to whether the enzyme also possesses single-stranded DNA and/or RNA 3′-5′ exonuclease activity. SAMHD1 was purified using three chromatography steps, over which the DNase activity was largely separated from the dNTPase activity, but the RNase activity persisted. Surprisingly, we found that catalytic and nucleotide activator site mutants of SAMHD1 with no dNTPase activity retained the exonuclease activities. Thus, the exonuclease activity cannot be associated with any known dNTP binding site. Monomeric SAMHD1 was found to bind preferentially to single-stranded RNA, while the tetrameric form required for dNTPase action bound weakly. ssRNA binding, but not ssDNA, induces higher-order oligomeric states that are distinct from the tetrameric form that binds dNTPs. We conclude that the trace exonuclease activities detected in SAMHD1 preparations arise from persistent contaminants that co-purify with SAMHD1 and not from the HD active site. An in vivo model is suggested where SAMHD1 alternates between the mutually exclusive functions of ssRNA binding and dNTP hydrolysis depending on dNTP pool levels and the presence of viral ssRNA.

Estrogen receptors (ERs) are expressed in leukocytes and in every ocular tissue. However, sex-specific differences and the role of estradiol in ocular inflammatory-reparative responses are not well understood. We found that female mice exhibited delayed corneal epithelial wound closure and attenuated polymorphonuclear (PMN) leukocyte responses, a phenotype recapitulated by estradiol treatment both in vivo (topically in male mice) and in vitro (corneal epithelial cell wound healing). The cornea expresses 15-lipoxygenase (15-LOX) and receptors for lipoxin A(4) (LXA(4)), which have been implicated in an intrinsic lipid circuit that regulates corneal inflammation and wound healing. Delayed epithelial wound healing correlated with lower expression of 15-LOX in the regenerated epithelium of female mice. Estradiol in vitro and in vivo down-regulated epithelial 15-LOX expression and LXA(4) formation, while estradiol abrogation of epithelial wound healing was completely reversed by treatment with LXA(4). More important, ERβ and ERα selectively regulated epithelial wound healing, PMN cell recruitment, and activity of the intrinsic 15-LOX/LXA(4) circuit. Our results demonstrate for the first time a sex-specific difference in the corneal reparative response, which is mediated by ERβ and ERα selective regulation of the epithelial and PMN 15-LOX/LXA(4) circuit. These findings may provide novel insights into the etiology of sex-specific ocular inflammatory diseases.