From the journals: JBC
Soothing COVID-19 symptoms with an over-the-counter medication. Crafting phophosproteomes with endosomal cAMP. Promoting stability through protein disorder. Read about papers on these and other topics in the Journal of Biological Chemistry.
Antacid may ease COVID-19 symptoms
Widely available vaccines have diminished the prevalence of severe illness and death due to COVID-19. However, the highly contagious delta variant has driven an increase in breakthrough infections among fully vaccinated individuals. While those vaccinated are still at lower risk for hospitalization and death, affordable and easily procurable therapeutic options remain limited for those suffering from the panoply of symptoms that accompany COVID-19. In a recent case series, the over-the-counter heartburn and ulcer medication famotidine, sold as Pepcid AC, rapidly relieved symptoms, including cough, fatigue, headaches and shortness of breath, in nonhospitalized COVID-19 patients, but researchers did not know the molecular and biological processes underlying these observations.
In recent work published in the Journal of Biological Chemistry, Rukmini Mukherjee of Goethe University and colleagues used biochemical, cellular and functional assays to assess the molecular actions of famotidine against SARS-CoV-2. While famotidine did not have an effect on viral replication or viral protease activities, the researchers showed that it does inhibit histamine-induced expression of toll-like receptor 3, or TLR3, a molecule that plays an important role in pathogen recognition and innate immune response.
Using Caco-2 cells, an immortalized line of human colorectal adenocarcinoma cells, infected with SARS-CoV-2, the scientists found that famotidine treatment reduced TLR3-dependent signaling processes that culminate in activation of interferon regulatory factor 3 and the NF-kappa B pathway, altering antiviral and inflammatory responses. SARS-CoV-2–infected cells treated with famotidine displayed reduced expression levels of the inflammatory mediators C–C motif chemokine ligand 2 and interleukin 6, which drive cytokine storms, a potentially life-threatening symptom of COVID-19.
Pharmacokinetic studies have shown that famotidine can reach concentrations in blood sufficient to antagonize histamine H2 receptors expressed in numerous cell types, including mast cells, neutrophils and eosinophils. Thus, these findings explain how famotidine may reduce histamine-induced inflammation and cytokine release, in turn improving outcomes in COVID-19 patients.
Endosomal cAMP alters the phosphoproteome
G protein–coupled receptors, or GPCRs, comprise the largest and most versatile class of cellular receptors, controlling nearly all essential mammalian physiological processes. Recently, researchers have realized that GPCRs can be activated to generate cyclic AMP, a second messenger that plays a critical role in GPCR-mediated signaling cascades, from endosomal membranes. Moreover, recent evidence suggests that this endosomal signaling may underlie important physiological and pharmacological phenomena, such as selective drug responses. Yet researchers’ knowledge of the functional consequences of this signaling is incomplete.
In a paper published in the Journal of Biological Chemistry, Nikoleta Tsvetanovaof the University of California, San Francisco, and colleagues combined an optogenetic approach for site-specific generation of cAMP with unbiased mass spectrometry–based analysis of the phosphoproteome. Using this method, the authors identified unique sites whose phosphorylation is changed in response to cAMP elevation. They also found a strong endosomal bias for a subset of proteins that are dephosphorylated in response to cAMP and implicate localized cAMP production in defining distinct phophoresponses.
Exploring a link between TBK1 and mTORC2
TANK-binding kinase 1, or TBK1, responds to infiltrating microbes by initiating cellular pathways critical for host innate immune defense. In previous work, Diane Fingar’s lab at the University of Michigan Medical School discovered that TBK1 phosphorylates the mechanistic target of rapamycin complex 1, or mTORC1, on serine 2159, or S2159, increasing mTORC1 signaling and mTORC1-mediated cell growth as well as the production of type 1 interferons such as interferon-beta. As part of this work, it was observed that knockout of TBK1 in mouse embryonic fibroblasts, or MEFs, led to a reduction in phosphorylation of Akt serine 473, an important metabolic kinase and target of phosphoinositide 3-kinase and an established target of mTORC2. However, researchers do not yet know the link between TBK1 and mTORC2.
In a recent study published in the Journal of Biological Chemistry, Aaron Seth Tooley and colleagues in the Fingar lab explored the existence of a direct functional relationship between TBK1 and mTORC2. Using MEFs lacking TBK1, wild-type MEFs and mice bearing an Mtor S2159A knock-in allele, the authors demonstrated that TBK1 activates mTORC2 directly to increase Akt phosphorylation. The researchers found that TBK1 and mTOR S2159 phosphorylation boosts mTOR-dependent phosphorylation of Akt in response to several growth factors. Immunoprecipitation experiments demonstrated TBK1–mTORC2 coimmunoprecipitation, and kinase assays showed that TBK1 and mTOR S2159 phosphorylation increase mTORC2 intrinsic catalytic activity.
These findings suggest the existence of crosstalk between TBK1 and mTOR and, as both TBK1 and mTOR contribute to tumorigenesis and metabolic disorders, future work may identify new pathways that contribute to disease pathology.
Disordered protein clusters promote stability
RNA-binding proteins play important roles in various cellular functions and contain abundant disordered protein regions, which account for 50% of the RNA-binding proteome. Among these disordered regions are electronegative clusters, or ENCs, which contain acidic residues and/or phosphorylation sites and whose abundance and length exceed other known repetitive sequences. Yet, despite this abundance, researchers know little about the functions of ENCs in RNA-binding proteins.
In a paper published in the Journal of Biological Chemistry, Steve Zaharias of the University of Alabama at Birminghamand colleagues investigate the impacts of ENCs on protein stability and RNA-binding affinity and specificity. Using the RNA-binding protein ribosomal biogenesis factor 15, Nop15, as a model, the researchers showed that Nop15 ENC increases protein stability, inhibits nonspecific RNA binding and regulates RNA binding via electrostatic interaction. The researchers grafted an ENC to another RNA-binding protein, Ser/Arg-rich splicing factor 3, and used RNA Bind-n-Seq — a high-throughput, cost-effective method for resolving sequence and secondary structure preferences of RNA-binding proteins — to show that the engineered ENC inhibits disparate RNA motifs differently, suggesting that one function of ENCs is to regulate RNA binding via electrostatic interaction.
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