The dNTPase activity of SAMHD1 is important for its suppression of innate immune responses in differentiated monocytic cells

Qin, Zhihua; Bonifati, Serena; Gelais, Corine St.; Li, Tai-Wei; Kim, Sun‐Hee; Antonucci, Jenna M.; Mahboubi, Bijan; Yount, Jacob S.; Xiong, Yong; Kim, Baek; Wu, Li

doi:10.1074/jbc.ra119.010360

“…Our studies were carried out in the absence of these viral components, which raises the possibility that viral proteins may also counteract SAMHD1-suppression of NF-κB in order to maintain HIV-1 replication. Furthermore, TRAF6 has been shown to regulate HIV-1 production in macrophages (57) Our current and previous results demonstrated that nuclear localization of SAMHD1 is dispensable for suppression of NF-κB activation in HEK293T cells and differentiated THP-1 or U937 monocytic cell lines (20). These observations support the notion that a cytoplasmic portion of SAMHD1 may specifically function to block proteins involved in NF-kB signaling, such as TRAF6/2, TAB3, and/or TAK1.…”

Section: Discussionsupporting

confidence: 80%

TRAF6 and TAK1 Contribute to SAMHD1-Mediated Negative Regulation of NF-κB Signaling

Espada

¹

,

Gelais

²

,

Bonifati

³

et al. 2021

J Virol

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Sterile alpha motif and HD-domain-containing protein 1 (SAMHD1) restricts HIV-1 replication by limiting the intracellular dNTP pool. SAMHD1 also suppresses the activation of NF-κB in response to viral infections and inflammatory stimuli. However, the mechanisms by which SAMHD1 negatively regulates this pathway remain unclear. Here we show that SAMHD1-mediated suppression of NF-κB activation is modulated by two key mediators of NF-κB signaling, tumor necrosis factor (TNF) receptor-associated factor 6 (TRAF6) and transforming growth factor-ß-activated kinase-1 (TAK1). We compared NF-κB activation stimulated by interleukin (IL)-1ß in monocytic THP-1 control and SAMHD1 knockout (KO) cells with and without partial TRAF6 knockdown (KD), or in cells treated with TAK1 inhibitors. Relative to control cells, IL-1ß-treated SAMHD1 KO cells showed increased phosphorylation of the inhibitor of NF-κB (IκBα), an indication of pathway activation, and elevated levels of TNF-α mRNA. Moreover, SAMHD1 KO combined with TRAF6 KD or pharmacological TAK1 inhibition reduced IκBα phosphorylation and TNF-α mRNA to the level of control cells. SAMHD1 KO cells infected with single-cycle HIV-1 showed elevated infection and TNF-α mRNA levels compared to control cells, and the effects were significantly reduced by TRAF6 KD or TAK1 inhibition. We further demonstrated that overexpressed SAMHD1 inhibited TRAF6-stimulated NF-κB reporter activity in HEK293T cells in a dose-dependent manner. SAMHD1 contains a nuclear localization signal (NLS), but an NLS-defective SAMHD1 exhibited a suppressive effect similar to the wild-type protein. Our data suggest that the TRAF6-TAK1 axis contributes to SAMHD1-mediated suppression of NF-κB activation and HIV-1 infection. Importance Cells respond to pathogen infection by activating a complex innate immune signaling pathway, which culminates in the activation of transcription factors and secretion of a family of functionally and genetically related cytokines. However, excessive immune activation may cause tissue damage and detrimental effects on the host. Therefore, in order to maintain host homeostasis, the innate immune response is tightly regulated during viral infection. We have reported SAMHD1 as a novel negative regulator of the innate immune response. Here, we provide new insights into SAMHD1-mediated negative regulation of the NF-κB pathway at the TRAF6-TAK1 checkpoint. We show that SAMHD1 inhibits TAK1 activation and TRAF6 signaling in response to proinflammatory stimuli. Interestingly, TRAF6 knockdown in SAMHD1-deficient cells significantly inhibited HIV-1 infection and activation of NF-κB induced by virus infection. Our research reveals a new negative regulatory mechanism by which SAMHD1 participates in the maintenance of cellular homeostasis during HIV-1 infection and inflammation.

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“…TAK1 activation by IL-1β or stimulation of host pathogen recognition receptors (PRRs) depends on the recruitment of the upstream factor TRAF6, which catalyzes the formation of K63-linked polyubiquitin chains (33). Using a NLS-defective SAMHD1 mutant (mNLS), we have demonstrated that SAMHD1 inhibits IL-1β-induced NF-κB activation independently of its NLS (20). To determine if SAMHD1 suppress NF-κB activation initiated through TRAF6, we used an NF-κB-luciferase reporter plasmid system.…”

Section: Resultsmentioning

confidence: 99%

“…The pcDNA3-GFP vector was previously described (59). The pCG-FLAG-HA plasmids (60) encoding the SAMHD1 mutant NLS (mNLS) and the P275A mutants were generated using a QuikChange Site-Directed Mutagenesis Kit (Agilent Technologies) according to the manufacturer’s protocol using the following primers: 5’-GAG CAG CCC TCC GCG GCT CCC GCT TGC GAT GAC AGC (mNLS) (20) and 5’-GGA ACA AAT TGT AGG AGC ACT TGA ATC ACC TGT C (SAMHD1 P275A). The pCG-FLAG-HA-SAMHD1 plasmid was a kind gift from Dr. J. Skowronski.…”

Section: Methodsmentioning

confidence: 99%

TRAF6 and TAK1 contribute to SAMHD1-mediated negative regulation of NF-κB signaling

Espada

¹

,

Gelais

²

,

Bonifati

³

et al. 2020

Preprint

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Sterile alpha motif and HD-domain-containing protein 1 (SAMHD1) restricts HIV-1 replication by limiting the intracellular dNTP pool. SAMHD1 also suppresses the activation of NF-κB in response to viral infections and inflammatory stimuli. However, the mechanisms by which SAMHD1 negatively regulates this pathway remain unclear. Here we show that SAMHD1-mediated suppression of NF-κB activation is modulated by two key mediators of NF-κB signaling, tumor necrosis factor (TNF) receptor-associated factor 6 (TRAF6) and transforming growth factor-β-activated kinase-1 (TAK1). We compared NF-κB activation stimulated by interleukin (IL)-1β in monocytic THP-1 control and SAMHD1 knockout (KO) cells with and without partial TRAF6 knockdown (KD), or in cells treated with TAK1 inhibitors. Relative to control cells, IL-1β-treated SAMHD1 KO cells showed increased phosphorylation of the inhibitor of NF-κB (IκBα), an indication of pathway activation, and elevated levels of TNF-α mRNA. Moreover, SAMHD1 KO combined with TRAF6 KD or pharmacological TAK1 inhibition reduced IκBα phosphorylation and TNF-α mRNA to the level of control cells. SAMHD1 KO cells infected with single-cycle HIV-1 showed elevated infection and TNF-α mRNA levels compared to control cells, and the effects were significantly reduced by TRAF6 KD or TAK1 inhibition. We further demonstrated that overexpressed SAMHD1 inhibited TRAF6-stimulated NF-κB reporter activity in HEK293T cells in a dose-dependent manner. SAMHD1 contains a nuclear localization signal (NLS), but an NLS-defective SAMHD1 exhibited a suppressive effect similar to the wild-type protein. Our data suggest that the TRAF6-TAK1 axis contributes to SAMHD1-mediated suppression of NF-κB activation and HIV-1 infection.

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“…As a nuclear localization sequence (NLS)-containing protein [46][47][48] (Figure 2), SAMHD1 has been suspected to maintain strict nuclear localization [9,49,50]. However, many studies have demonstrated that SAMHD1 can be partially cytosolic [46,51,52]-maintaining relatively equal nuclear and cytosolic populations in some cell types [53,54]-and that its localization varies due to the cellular environment. Upon oxidative stress, SAMHD1 was found to migrate from the nucleus to the cytoplasm, only to return back to the nucleus one hour after stimulation [43].…”

Section: Samhd1 Protein Contains An Nls and Is Expressed In A Varietymentioning

confidence: 99%

SAMHD1 Functions and Human Diseases

Coggins

¹

,

Mahboubi

²

,

Schinazi

³

et al. 2020

Viruses

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Deoxynucleoside triphosphate (dNTP) molecules are essential for the replication and maintenance of genomic information in both cells and a variety of viral pathogens. While the process of dNTP biosynthesis by cellular enzymes, such as ribonucleotide reductase (RNR) and thymidine kinase (TK), has been extensively investigated, a negative regulatory mechanism of dNTP pools was recently found to involve sterile alpha motif (SAM) domain and histidine-aspartate (HD) domain-containing protein 1, SAMHD1. When active, dNTP triphosphohydrolase activity of SAMHD1 degrades dNTPs into their 2′-deoxynucleoside (dN) and triphosphate subparts, steadily depleting intercellular dNTP pools. The differential expression levels and activation states of SAMHD1 in various cell types contributes to unique dNTP pools that either aid (i.e., dividing T cells) or restrict (i.e., nondividing macrophages) viral replication that consumes cellular dNTPs. Genetic mutations in SAMHD1 induce a rare inflammatory encephalopathy called Aicardi–Goutières syndrome (AGS), which phenotypically resembles viral infection. Recent publications have identified diverse roles for SAMHD1 in double-stranded break repair, genome stability, and the replication stress response through interferon signaling. Finally, a series of SAMHD1 mutations were also reported in various cancer cell types while why SAMHD1 is mutated in these cancer cells remains to investigated. Here, we reviewed a series of studies that have begun illuminating the highly diverse roles of SAMHD1 in virology, immunology, and cancer biology.

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The dNTPase activity of SAMHD1 is important for its suppression of innate immune responses in differentiated monocytic cells

Cited by 14 publications

References 44 publications

TRAF6 and TAK1 Contribute to SAMHD1-Mediated Negative Regulation of NF-κB Signaling

TRAF6 and TAK1 Contribute to SAMHD1-Mediated Negative Regulation of NF-κB Signaling

TRAF6 and TAK1 contribute to SAMHD1-mediated negative regulation of NF-κB signaling

SAMHD1 Functions and Human Diseases

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