Biology subjects

Schott, K.

Publications and source records attributed to Schott, K..

4 recordsLinked to original sources

bioRxiv2023

Gene editing of SAMHD1 in macrophage-like cells reveals complex relationships between SAMHD1 phospho-regulation, HIV-1 restriction and cellular dNTP levels

Sterile motif (SAM) and HD domain-containing protein 1 (SAMHD1) is a dNTP triphosphate triphosphohydrolase (dNTPase) and a potent restriction factor for immunodeficiency virus 1 (HIV-1), active in myeloid and resting CD4+ T cells. The anti-viral activity of SAMHD1 is regulated by dephosphorylation of the residue T592. However, the impact of T592 phosphorylation on dNTPase activity is still under debate. Whether additional cellular functions of SAMHD1 impact anti-viral restriction is not completely understood. We report BLaER1 cells as a novel human macrophage HIV-1 infection model combined with CRISPR/Cas9 knock-in (KI) introducing specific mutations into the SAMHD1 locus to study mutations in a physiological context. Transdifferentiated BLaER1 cells harbor active dephosphorylated SAMHD1 that blocks HIV-1 reporter virus infection. As expected, homozygous T592E mutation, but not T592A, relieved a block to HIV-1 reverse transcription. Co-delivery of VLP-Vpx to SAMHD1 T592E KI mutant cells did not further enhance HIV-1 infection indicating the absence of an additional SAMHD1-mediated antiviral activity independent of T592 de-phosphorylation. T592E KI cells retained dNTP levels similar to WT cells indicating uncoupling of anti-viral and dNTPase activity of SAMHD1. The integrity of the catalytic site in SAMHD1 was critical for anti-viral activity, yet poor correlation of HIV-1 restriction and global cellular dNTP levels was observed in cells harboring catalytic core mutations. Together, we emphasize the complexity of the relationship between HIV-1 restriction, SAMHD1 enzymatic function and T592 phospho-regulation and provide novel tools for investigation in an endogenous and physiological context. ImportanceWe introduce BLaER1 cells as an alternative myeloid cell model in combination with CRISPR/Cas9-mediated gene editing to study the influence of SAMHD1 T592 Mophosphorylation on anti-viral restriction and the control of cellular dNTP levels in an endogenous, physiological relevant context. Proper understanding of the mechanism of the anti-viral function of SAMHD1 will provide attractive strategies aiming at selectively manipulating SAMHD1 without affecting other cellular functions. Even more, our toolkit may inspire further genetic analysis and investigation of restriction factors inhibiting retroviruses, their cellular function and regulation, leading to a deeper understanding of intrinsic anti-viral immunity.

microbiology↗

bioRxiv2023

Epigenetic changes and serotype-specific interferon-responses of lung epithelial cells in late post-influenza pneumococcal pneumonia

Pneumococcal infection following influenza A virus (IAV) pneumonia is a synergistic complication with high mortality. IAV modulates host antibacterial responses and invasiveness of pneumococcal serotypes and is an important pathogenic factor 2. Yet, serotype-specifc immediate-early responses of the IAV-perturbed alveolar epithelium have not been adressed. We analyzed gene transcription in alveolar type II epithelial cells (AECII) from mice infected with IAV and/or one of three S. pneumoniae (S.pn.) serotypes of varying invasiveness (4 > 7F > 19F). IAV, 14 days post infection, rendered the lung susceptible to invasive S.pn. infection with serotype 4 and the mildly invasive 7F but not 19F. Only 7F secondary infection induced exacerbated cytokine/chemokine responses. IAV/7F infection induced superior protein expression of type I and II interferons, exceeding that in IAV/serotype 4 infection. Inference of a scale-free-like ARACNE gene co-expression network revealed interferon-response network modules. Network-mapping unfolded S.pn. serotype-specific transcriptional network responses/usage. Secondary S.pn. infection abrogated the IAV-induced pneumocyte proliferative configuration and IAV infection rendered the transcriptional response to 7F comparable to that of serotype 4. This related to network genes correlating with the expression of two master regulators of interferon responses: Irf7 and Stat1. Epigenetic ATAC-seq analysis of AECII in resolved IAV infection identified enhanced expression of ARACNE network genes Hist1h2bf, Igtp, Mki67, Rasl10b, H2-Q6 and H2-Q7 to be associated with increased chromatin accessability at promoter regions. We show that AECII retain a sustained IAV-associated transcriptional configuration with epigenetic involvement that serotype-specifically affects proliferation and intensifies the AECII transcriptional response, mainly to interferons, in S.pn. infection.

immunology↗

bioRxiv2023

Unplugging DI-DIV and DII-DIII lateral fenestrations of NALCN reveals unexpected pharmacology

The sodium (Na+) leak channel (NALCN) is a member of the four-domain voltage-gated cation channel family that includes the prototypical voltage-gated sodium and calcium channels (NaVs and CaVs, respectively). Unlike NaVs and CaVs, which have four lateral fenestrations that serve as routes for lipophilic compounds to enter the central cavity to modulate channel function, NALCN has bulky residues (W311, L588, M1145 and Y1436) that block these openings. Structural data suggest that oc-cluded lateral fenestrations underlie the pharmacological resistance of NALCN to lipophilic compounds, but functional evidence is lacking. To test this hypothesis, we unplugged the fenestrations of NALCN by substituting the four aforementioned resi-dues with alanine (AAAA) and compared the effects of NaV, CaV and NALCN block-ers on both wild-type (WT) and AAAA channels. Most compounds behaved in a simi-lar manner on both channels, but phenytoin and 2-aminoethoxydiphenyl borate (2-APB) elicited additional, distinct responses on AAAA channels. Further experiments using single alanine mutants revealed that phenytoin and 2-APB enter the inner cav-ity through distinct fenestrations, implying structural specificity to their modes of ac-cess. Using a combination of computational and functional approaches, we identified amino acid residues critical for 2-APB activity, supporting the existence of drug bind-ing site(s) within the pore region. Intrigued by the activity of 2-APB and its ana-logues, we tested additional compounds containing the diphenylmethane/amine moiety on WT channels. We identified compounds from existing clinically used drugs that exhibited diverse activity, thus expanding the pharmacological toolbox for NALCN. While the low potencies of active compounds reiterate the resistance of NALCN to pharmacological targeting, our findings lay the foundation for rational drug design to develop NALCN modulators with refined properties. Significance statementThe sodium leak channel (NALCN) is essential for survival: mutations cause life-threatening developmental disorders in humans. However, no treatment is currently available due to the resistance of NALCN to pharmacological targeting. One likely reason is that the lateral fenestrations, a common route for clinically used drugs to enter and block related ion channels, are occluded in NALCN. Using a combination of computational and functional approaches, we unplugged the fenestrations of NALCN which led us to the first molecularly defined drug binding site within the pore region. Besides that, we also identified additional NALCN modulators from existing clinically used therapeutics, thus expanding the pharmacological toolbox for this leak channel.

pharmacology and toxicology↗

bioRxiv2022

CRISPR/Cas9 knock-in strategy to evaluate phospho-regulation of SAMHD1

Sterile motif (SAM) and HD domain-containing protein 1 (SAMHD1) is a potent restriction factor for immunodeficiency virus 1 (HIV-1), active in myeloid and resting CD4+ T cells. As a dNTP triphosphate triphosphohydrolase (dNTPase), SAMHD1 is proposed to limit cellular dNTP levels correlating with inhibition of HIV-1 reverse transcription. The anti-viral activity of SAMHD1 is regulated by dephosphorylation of the residue T592. However, the impact of T592 phosphorylation on dNTPase activity is still under debate. Whether additional cellular functions of SAMHD1 impact anti-viral restriction is also not completely understood. We use BlaER1 cells as a novel human macrophage transdifferentiation model combined with CRISPR/Cas9 knock-in (KI) to study SAMHD1 mutations in a physiological context. Transdifferentiated BlaER1 cells, resembling primary human macrophages, harbor active dephosphorylated SAMHD1 that blocks HIV-1 reporter virus infection. Co-delivery of Vpx or CRISPR/Cas9-mediated SAMHD1 knock-out relieves the block to HIV-1. Using CRISPR/Cas9-mediated homologous recombination, we introduced specific mutations into the genomic SAMHD1 locus. Homozygous T592E mutation, but not T592A, leads to loss of HIV-1 restriction, confirming the role of T592 dephosphorylation in the regulation of anti-viral activity. However, T592E KI cells retain wild type dNTP levels, suggesting the antiviral state might not only rely on dNTP depletion. In conclusion, the role of the T592 phospho-site for anti-viral restriction was confirmed in an endogenous physiological context. Importantly, loss of restriction in T592E mutant cells does not correlate with increased dNTP levels, indicating that the regulation of anti-viral and dNTPase activity of SAMHD1 might be uncoupled. ImportanceSterile motif (SAM) and HD domain-containing protein 1 (SAMHD1) is a potent anti-viral restriction factor, active against a broad range of DNA viruses and retroviruses. In myeloid and resting CD4+ T cells, SAMHD1 blocks reverse transcription of immunodeficiency virus 1 (HIV-1), not only inhibiting viral replication in these cell types, but also limiting the availability of reverse transcription products for innate sensing of HIV-1. Manipulating SAMHD1 activity could be an attractive approach to improve HIV-1 therapy or vaccination strategies. Anti-viral activity is strictly dependent on dephosphorylation of SAMHD1 residue T592, however the mechanistic consequence of T592 phosphorylation is still unclear. Here, we use BlaER1 cells as an alternative myeloid cell model in combination with CRISPR/Cas9-mediated KI to study the influence of SAMHD1 T592 phosphorylation on anti-viral restriction and control of cellular dNTP levels in an endogenous context. By using this novel approach, we were able to genetically uncouple SAMHD1s anti-viral and dNTPase activity with regard to regulation by T592 phosphorylation. This suggests that SAMHD1 dNTPase activity may not exclusively be responsible for the anti-lentiviral activity of SAMHD1 in myeloid cells. In addition, our toolkit may inspire further genetic analysis and investigation of SAMHD1-mediated restriction, as wells as its cellular function and regulation, leading to a deeper understanding of SAMHD1 and HIV-1 biology.

microbiology↗