Identification of herbal teas and their compounds eliciting antiviral activity against SARS-CoV-2 in vitro

Background: The SARS-CoV-2/COVID-19 pandemic has inflicted medical and socioeconomic havoc, and despite the current availability of vaccines and broad implementation of vaccination programs, more easily accessible and cost-effective acute treatment options preventing morbidity and mortality are urgently needed. Herbal teas have historically and recurrently been applied as self-medication for prophylaxis, therapy, and symptom alleviation in diverse diseases, including those caused by respiratory viruses, and have provided sources of natural products as basis for the development of therapeutic agents. To identify affordable, ubiquitously available, and effective treatments, we tested herbs consumed worldwide as herbal teas regarding their antiviral activity against SARS-CoV-2. Results: Aqueous infusions prepared by boiling leaves of the Lamiaceae perilla and sage elicit potent and sustained antiviral activity against SARS-CoV-2 when applied after infection as well as prior to infection of cells. The herbal infusions exerted in vitro antiviral effects comparable to interferon-β and remdesivir but outperformed convalescent sera and interferon-α2 upon short-term treatment early after infection. Based on protein fractionation analyses, we identified caffeic acid, perilla aldehyde, and perillyl alcohol as antiviral compounds. Global mass spectrometry (MS) analyses performed comparatively in two different cell culture infection models revealed changes of the proteome upon treatment with herbal infusions and provided insights into the mode of action. As inferred by the MS data, induction of heme oxygenase 1 (HMOX-1) was confirmed as effector mechanism by the antiviral activity of the HMOX-1-inducing compounds sulforaphane and fraxetin. Conclusions: In conclusion, herbal teas based on perilla and sage exhibit antiviral activity against SARS-CoV-2 including variants of concern such as Alpha, Beta, Delta, and Omicron, and we identified HMOX-1 as potential therapeutic target. Given that perilla and sage have been suggested as treatment options for various diseases, our dataset may constitute a valuable resource also for future research beyond virology

Impairment of angiogenesis by fatty acid synthase inhibition Involves mTOR malonylation

The role of fatty acid synthesis in endothelial cells (ECs) remains incompletely characterized. We report that fatty acid synthase knockdown (FASNKD) in ECs impedes vessel sprouting by reducing proliferation. Endothelial loss of FASN impaired angiogenesis in vivo, while FASN blockade reduced pathological ocular neovascularization, at >10-fold lower doses than used for anti-cancer treatment. Impaired angiogenesis was not due to energy stress, redox imbalance, or palmitate depletion. Rather, FASNKD elevated malonyl-CoA levels, causing malonylation (a post-translational modification) of mTOR at lysine 1218 (K1218). mTOR K-1218 malonylation impaired mTOR complex 1 (mTORC1) kinase activity, thereby reducing phosphorylation of downstream targets (p70S6K/4EBP1). Silencing acetyl-CoA carboxylase 1 (an enzyme producing malonyl-CoA) normalized malonyl-CoA levels and reactivated mTOR in FASNKD ECs. Mutagenesis unveiled the importance of mTOR K1218 malonylation for angiogenesis. This study unveils a novel role of FASN in metabolite signaling that contributes to explaining the anti-angiogenic effect of FASN blockade

Current strategies and findings in clinically relevant post-translational modification-specific proteomics

Mass spectrometry-based proteomics has considerably extended our knowledge about the occurrence and dynamics of protein post-translational modifications (PTMs). So far, quantitative proteomics has been mainly used to study PTM regulation in cell culture models, providing new insights into the role of aberrant PTM patterns in human disease. However, continuous technological and methodical developments have paved the way for an increasing number of PTM-specific proteomic studies using clinical samples, often limited in sample amount. Thus, quantitative proteomics holds a great potential to discover, validate and accurately quantify biomarkers in body fluids and primary tissues. A major effort will be to improve the complete integration of robust but sensitive proteomics technology to clinical environments. Here, we discuss PTMs that are relevant for clinical research, with a focus on phosphorylation, glycosylation and proteolytic cleavage; furthermore, we give an overview on the current developments and novel findings in mass spectrometry-based PTM research

Highly Sensitive Phosphoproteomics by Tailoring Solid-Phase Extraction to Electrostatic Repulsion-Hydrophilic Interaction Chromatography

In the past decade, several strategies for comprehensive phosphoproteome analysis have been introduced. Most of them combine different phosphopeptide enrichment techniques and require starting material in the milligram range, as a consequence of their insufficient sensitivity. This limitation impairs the applicability of phosphoproteomics to a wide variety of clinical research, where sample material is highly limited. Here we introduce a highly sensitive and easy-to-establish 2D bottom-up strategy for microgram-scale phosphoproteomics, based on electrostatic repulsion–hydrophilic interaction chromatography (ERLIC), a simple solid-phase extraction step by strong cation exchange (SCX) or reversed phase (RP), and LC-MS analysis. With only 100 μg of tryptic digested, nonstimulated HeLa protein and 45 h of LC-MS analysis time, we identified ≥7500 nonredundant and highly confident phosphorylation sites (per replicate). We assigned all phosphorylation sites to 3013 phosphoproteins, covering the entire dynamic range from 10⁷ down to a few copies per cell. Compared to affinity-based-enrichment methods using Ti⁴⁺, our ERLIC-based strategy enriched considerably longer and more acidic phosphopeptides and consequently, we identified 327 phosphorylated C-terminal peptides. The simplicity and high sensitivity of ERLIC-SCX/RP-LC-MS render its future promising for microgram-scale-phosphoproteomics in biological, biomedical, and clinical research

Highly Sensitive Phosphoproteomics by Tailoring Solid-Phase Extraction to Electrostatic Repulsion-Hydrophilic Interaction Chromatography

In the past decade, several strategies for comprehensive phosphoproteome analysis have been introduced. Most of them combine different phosphopeptide enrichment techniques and require starting material in the milligram range, as a consequence of their insufficient sensitivity. This limitation impairs the applicability of phosphoproteomics to a wide variety of clinical research, where sample material is highly limited. Here we introduce a highly sensitive and easy-to-establish 2D bottom-up strategy for microgram-scale phosphoproteomics, based on electrostatic repulsion–hydrophilic interaction chromatography (ERLIC), a simple solid-phase extraction step by strong cation exchange (SCX) or reversed phase (RP), and LC-MS analysis. With only 100 μg of tryptic digested, nonstimulated HeLa protein and 45 h of LC-MS analysis time, we identified ≥7500 nonredundant and highly confident phosphorylation sites (per replicate). We assigned all phosphorylation sites to 3013 phosphoproteins, covering the entire dynamic range from 10⁷ down to a few copies per cell. Compared to affinity-based-enrichment methods using Ti⁴⁺, our ERLIC-based strategy enriched considerably longer and more acidic phosphopeptides and consequently, we identified 327 phosphorylated C-terminal peptides. The simplicity and high sensitivity of ERLIC-SCX/RP-LC-MS render its future promising for microgram-scale-phosphoproteomics in biological, biomedical, and clinical research

Activation of E2F-dependent transcription by the mouse cytomegalovirus M117 protein affects the viral host range.

Cytomegaloviruses (CMVs) have a highly restricted host range as they replicate only in cells of their own or closely related species. To date, the molecular mechanisms underlying the CMV host restriction remain poorly understood. However, it has been shown that mouse cytomegalovirus (MCMV) can be adapted to human cells and that adaptation goes along with adaptive mutations in several viral genes. In this study, we identify MCMV M117 as a novel host range determinant. Mutations in this gene enable the virus to cross the species barrier and replicate in human RPE-1 cells. We show that the M117 protein is expressed with early kinetics, localizes to viral replication compartments, and contributes to the inhibition of cellular DNA synthesis. Mechanistically, M117 interacts with members of the E2F transcription factor family and induces E2F target gene expression in murine and human cells. While the N-terminal part of M117 mediates E2F interaction, the C-terminal part mediates self-interaction. Both parts are required for the activation of E2F-dependent transcription. We further show that M117 is dispensable for viral replication in cultured mouse fibroblasts and endothelial cells, but is required for colonization of mouse salivary glands in vivo. Conversely, inactivation of M117 or pharmacological inhibition of E2F facilitates MCMV replication in human RPE-1 cells, whereas replacement of M117 by adenovirus E4orf6/7, a known E2F activator, prevents it. These results indicate that E2F activation is detrimental for MCMV replication in human cells. In summary, this study identifies MCMV M117 as a novel E2F activator that functions as a host range determinant by precluding MCMV replication in human cells

Cyclin Y Is Expressed in Platelets and Modulates Integrin Outside-in Signaling

Diabetes is associated with platelet hyper-reactivity and enhanced risk of thrombosis development. Here we compared protein expression in platelets from healthy donors and diabetic patients to identify differentially expressed proteins and their possible function in platelet activation. Mass spectrometry analyses identified cyclin Y (CCNY) in platelets and its reduced expression in platelets from diabetic patients, a phenomenon that could be attributed to the increased activity of calpains. To determine the role of CCNY in platelets, mice globally lacking the protein were studied. CCNY-/- mice demonstrated lower numbers of circulating platelets but platelet responsiveness to thrombin and a thromboxane A2 analogue were comparable with that of wild-type mice, as was agonist-induced α and dense granule secretion. CCNY-deficient platelets demonstrated enhanced adhesion to fibronectin and collagen as well as an attenuated spreading and clot retraction, indicating an alteration in “outside in” integrin signalling. This phenotype was accompanied by a significant reduction in the agonist-induced tyrosine phosphorylation of β3 integrin. Taken together we have shown that CCNY is present in anucleated platelets where it is involved in the regulation of integrin-mediated outside in signalling associated with thrombin stimulation

Quantifying Missing (Phospho)Proteome Regions with the Broad-Specificity Protease Subtilisin

Despite huge efforts to map the human proteome using mass spectrometry the overall sequence coverage achieved to date is still below 50%. Reasons for missing areas of the proteome comprise protease-resistant domains including the lack/excess of enzymatic cleavage sites, nonunique peptide sequences, impaired peptide ionization/separation and low expression levels. To access novel areas of the proteome the beneficial use of enzymes complementary to trypsin, such as Glu-C, Asp-N, Lys-N, Arg-C, LysargiNase has been reported. Here, we present how the broad-specificity protease subtilisin enables mapping of previously hidden areas of the proteome. We systematically evaluated its digestion efficiency and reproducibility and compared it to the gold standard in the field, trypsin. Notably, subtilisin allows reproducible near-complete digestion of cells lysates in 1–5 min. As expected from its broad specificity the generation of overlapping peptide sequences reduces the number of identified proteins compared to trypsin (8363 vs 6807; 1% protein FDR). However, subtilisin considerably improved the coverage of missing and particularly proline-rich areas of the proteome. Along 14 628 high confidence phosphorylation sites identified in total, only 33% were shared between both enzymes, while 37% were exclusive to subtilisin. Notably, 926 of these were not even accessible by additional in silico digestion with either Asp-N, Arg-C, Glu-C, Lys-C, or Lys-N. Thus, subtilisin might be particularly beneficial for system-wide profiling of post-translational modification sites. Finally, we demonstrate that subtilisin can be used for reporter-ion based in-depth quantification, providing a precision comparable to trypsindespite broad specificity and fast digestion that may increase technical variance

A sensitive and simple targeted proteomics approach to quantify transcription factor and membrane proteins of the unfolded protein response pathway in glioblastoma cells

Many cellular events are driven by changes in protein expression, measurable by mass spectrometry or antibody-based assays. However, using conventional technology, the analysis of transcription factor or membrane receptor expression is often limited by an insufficient sensitivity and specificity. To overcome this limitation, we have developed a high-resolution targeted proteomics strategy, which allows quantification down to the lower attomol range in a straightforward way without any prior enrichment or fractionation approaches. The method applies isotope-labeled peptide standards for quantification of the protein of interest. As proof of principle, we applied the improved workflow to proteins of the unfolded protein response (UPR), a signaling pathway of great clinical importance, and could for the first time detect and quantify all major UPR receptors, transducers and effectors that are not readily detectable via antibody-based-, SRM- or conventional PRM assays. As transcription and translation is central to the regulation of UPR, quantification and determination of protein copy numbers in the cell is important for our understanding of the signaling process as well as how pharmacologic modulation of these pathways impacts on the signaling. These questions can be answered using our newly established workflow as exemplified in an experiment using UPR perturbation in a glioblastoma cell lines

Simple, scalable and ultra-sensitive tip-based identification of protease substrates

Proteases are in the center of many diseases and consequently proteases and their substrates are important drug targets as represented by an estimated 5-10% of all drugs under development. Mass spectrometry has been an indispensable tool for the discovery of novel protease substrates, particularly through the proteome-scale enrichment of so-called N-terminal peptides representing endogenous protein N-termini. Methods such as COmbined FRActional DIagonal Chromatography (COFRADIC) and later Terminal Amine Isotopic Labeling of Substrates (TAILS) have revealed numerous insights into protease substrates and consensus motifs. We present an alternative and simple protocol for N-terminal peptide enrichment, based on Charge-based FRActional DIagonal Chromatography (ChaFRADIC) and requiring only well-established protein chemistry and a pipette tip. Using iTRAQ-8plex we quantified on average 2073±52 unique N-terminal peptides from only 4.3 μg per sample/channel, allowing the identification of proteolytic targets and consensus motifs. This high sensitivity may even allow working with clinical samples such as needle biopsies in the future. We applied our method to study the dynamics of staurosporine-induced apoptosis. Our data demonstrate an orchestrated regulation of specific pathways after 1.5 h, 3 h and 6 h of treatment, with many important players of homeostasis targeted already after 1.5 h. We additionally observed an early multi-level modulation of the splicing machinery both by proteolysis and phosphorylation. This may reflect the known role of alternative splicing variants for a variety of apoptotic genes which seems to be a driving-force of staurosporine-induced apoptosis.status: publishe