Absin Product abs50001 Annexin V-FITC/PI Apoptosis Detection Kit Joins the Family of Cell Metabolism (IF 27.287)

1. Overview

Host cells co-regulate their metabolism through innate immune activation to combat viral infections. This study reveals the critical role of serine metabolism in blocking antiviral innate immunity and demonstrates that serine metabolism deficiency reduces SAM-mediated H3K27me3 and promotes ATP6V0d2 expression, thereby enhancing YAP lysosomal degradation and virus-induced IFN-β production.

2. Research Background

Innate immunity is the first important line of defense for the host against microbial infections. Host cells recognize viral RNA and DNA through different types of pattern recognition receptors (PRRs), including Toll-like receptors (TLRs), retinoic acid-inducible gene I (RIG-I)-like receptors, and cytoplasmic sensors for double-stranded DNA. PRRs then recruit adaptor proteins such as TRIF, MAVS, and STING to activate TBK1 and/or IKKε kinases. TBK1/IKKε phosphorylate IRF3 and nuclear factor κB (NF-κB) to induce their nuclear translocation and subsequent transcription of type I interferons (IFNs) (including IFN-α and IFN-β) and pro-inflammatory cytokines. Upon binding to IFN receptors, type I IFNs activate the JAK-STAT signal and promote the expression of IFN-stimulated genes to combat viral infections and coordinate adaptive immunity.

In de novo synthesis, glycolysis-derived 3-PG can be ultimately converted to serine through a series of enzymatic reactions, the first key step of which is catalyzed by phosphoglycerate dehydrogenase (PHGDH). Serine promotes one-carbon metabolism, including an interconnected network of metabolic pathways that facilitate the transfer of one-carbon units for the biosynthesis of nucleotides, S-adenosylmethionine (SAM), reduced nicotinamide adenine dinucleotide phosphate, and glutathione (GSH). Serine-derived one-carbon metabolism supports nucleotide synthesis for cancer and T cell proliferation. In addition, serine promotes lipopolysaccharide (LPS)-mediated interleukin (IL)-1β production in macrophages through one-carbon metabolism-facilitated GSH synthesis or SAM-mediated histone methylation. However, it remains unclear whether the PHGDH-mediated serine synthesis pathway (SSP) or exogenous serine is involved in antiviral innate immunity.

Vacuolar H+-adenosine triphosphatase (V-ATPase) is an ATP-dependent proton pump composed of a peripheral V1 domain that hydrolyzes ATP and an integral domain that transports protons. V-ATPase is localized on various cell membranes and acidifies intracellular compartments, including endosomes and lysosomes. V-ATPase or its subunit ATP6V0d2 is involved in regulating multiple biological processes, including protein degradation, promotion of viral fusion, or elimination of bacterial infections. However, the role of V-ATPase in antiviral immunity is currently unclear.

The Hippo pathway plays a key role in organ size control and tissue homeostasis. When this pathway is activated, Lats1/2 kinases phosphorylate YAP/TAZ, retaining them in the cytoplasm for ubiquitination and degradation. Otherwise, YAP/TAZ enter the nucleus and bind to TEAD family transcription factors to induce gene transcription. A recent study showed that after viral infection, YAP can prevent the dimerization of IRF3 and prevent IRF3 translocation to the nucleus (Wang et al., 2017). Meanwhile, virus-activated IKKε can phosphorylate YAP, promoting its lysosomal degradation and alleviating YAP-mediated inhibition of IFN-β production (Wang et al., 2017). Serum starvation can inactivate YAP/TAZ through Lats1/2 kinases, thereby attenuating YAP-mediated inhibition of TBK1 and enhancing the antiviral IFN-β response. However, it is currently unclear whether granular metabolic pathways can communicate with the Hippo YAP pathway to regulate antiviral innate immunity.

3. Experimental Methods Used in the Study

•         Plasmid and Transfection

•         Enzyme-Linked Immunosorbent Assay (ELISA) and Serine Measurement

•         Protein Isolation and Western Blot Analysis

•         RNA Extraction, RT-PCR, and RNA Sequencing Analysis

•         In Vivo Viral Infection

•         Lung Histology

•         Viral Infection and Plaque Assay

•         Luciferase Reporter Assay

•         LC-MS Analysis of Metabolites

•         Chromatin Immunoprecipitation (ChIP)

•         Flow Cytometry

4. Research Sections of the Article

•         PHGDH Negatively Regulates IFN-β Signaling by Inhibiting the TBK1-IRF3 Axis

•         Serine Metabolism Antagonizes Virus-Induced IFN-β Production

•         Serine Metabolism Deficiency Protects Mice from Viral Infection

•         SAM Derived from Serine Metabolism Impairs IFN-β Production

•         PHGDH Partially Inhibits IFN-β Production by Downregulating ATP6V0d2

•         ATP6V0d2 Induces IFN-β Production by Promoting Lysosomal Degradation of YAP During Viral Infection

5. Research Conclusions

The impact of serine metabolism on antiviral innate immunity. First, we show that the de novo serine biosynthesis pathway is significantly downregulated in mouse macrophages and HEK293T cells after viral infection. Targeting the activity of PHGDH, a key enzyme in the SSP, through genetic regulation or treatment with the inhibitor CBR-5884, potently enhances IFN-β-mediated antiviral innate immunity in vitro and in vivo. In addition, exogenous serine and glycine restriction also alleviates viral infection by enhancing IFN-β in vitro and in vivo. Mechanistically, we found that inhibition of endogenous and exogenous serine metabolism promotes the expression of ATP6V0d2 by reducing SAM-mediated H3K27me3. ATP6V0d2 targets YAP for lysosomal degradation to attenuate YAP-mediated inhibition of the TBK1-IRF3 axis and enhance IFN-β-mediated antiviral innate immunity.

In summary, our findings not only clarify a previously unknown mechanism by which phosphoglycerate dehydrogenase (PHGDH)/serine plays a key role in controlling antiviral signaling pathways but also propose manipulating serine metabolism as a potential antiviral therapeutic approach.

6. Flow Cytometry Used in the Article

Peritoneal macrophages from Phgdhfl/flLyz2 Cre+ and Phgdhfl/flLyz2 Cre- mice were collected and filtered to prepare single-cell suspensions. To detect the differentiation and number of peritoneal macrophages in the two mouse genotypes, cells were washed with ice-cold PBS containing 1% FBS, incubated with APC anti-mouse CD11b antibody and PE/Cyanine5 anti-mouse F4/80 antibody on ice for 15 minutes, and then subjected to FACS using a BD LSRFortessa flow cytometer (BD Biosciences). Apoptosis detection of SeV-infected peritoneal macrophages was performed with Propidium Iodide and Annexin-V-FITC (Absin). Data were acquired by flow cytometry and analyzed using FlowJo (Tree Star).

Apoptosis of SeV-infected mouse PMs at 6h was analyzed by PI and Annexin V staining.

7. About Absin's abs50001 Annexin V-FITC/PI Apoptosis Detection Kit

The abs50001 Annexin V-FITC/PI Apoptosis Detection Kit from Absin is a high-quality research tool widely used in apoptosis detection experiments. Its outstanding performance has been verified in important studies published in top journals such as Cell Metabolism (IF 27.287), providing reliable support for researchers in the field of immunology and virology.

This kit uses Annexin V-FITC and PI double staining to accurately distinguish early apoptotic cells, late apoptotic cells, and necrotic cells. Annexin V can specifically bind to phosphatidylserine (PS) exposed on the surface of early apoptotic cells, while PI cannot penetrate the intact cell membrane of living cells and early apoptotic cells but can enter late apoptotic cells and necrotic cells to stain nuclear DNA. Through flow cytometry analysis, researchers can intuitively obtain the apoptotic status of cells, which is crucial for studying the regulatory mechanism of cell apoptosis and the interaction between viruses and host cells.

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9. About AN BIO PTE. LTD.

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10. Disclaimer

This article is compiled and interpreted by AI based on the original work with DOI: 10.1002/advs.202413562. All intellectual property rights (such as images, data) of the original publication belong to the journal and the research team. For any infringement, please contact us immediately and we will take prompt action.

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