Mitochondrial N-formyl methionine peptides contribute to exaggerated neutrophil activation in patients with COVID-19.

Neutrophil dysregulation is well established in COVID-19. However, factors contributing to neutrophil activation in COVID-19 are not clear. We assessed if N-formyl methionine (fMet) contributes to neutrophil activation in COVID-19. Elevated levels of calprotectin, neutrophil extracellular traps (NETs) and fMet were observed in COVID-19 patients (n = 68), particularly in critically ill patients, as compared to HC (n = 19, p < 0.0001). Of note, the levels of NETs were higher in ICU patients with COVID-19 than in ICU patients without COVID-19 (p < 0.05), suggesting a prominent contribution of NETs in COVID-19. Additionally, plasma from COVID-19 patients with mild and moderate/severe symptoms induced in vitro neutrophil activation through fMet/FPR1 (formyl peptide receptor-1) dependent mechanisms (p < 0.0001). fMet levels correlated with calprotectin levels validating fMet-mediated neutrophil activation in COVID-19 patients (r = 0.60, p = 0.0007). Our data indicate that fMet is an important factor contributing to neutrophil activation in COVID-19 disease and may represent a potential target for therapeutic intervention.

Chemo-Enzymatic Modification of the 5' Cap To Study mRNAs.

The central dogma of molecular biology hinges on messenger RNA (mRNA), which presents a blueprint of the genetic information encoded in the DNA and serves as a template for translation into proteins. In addition to its fundamental importance in basic research, this class of biomolecules has recently become the first approved Covid vaccine, underscoring its utility in medical applications.Eukaryotic mRNA is heavily processed, including the 5' cap as the primary hallmark. This 5' cap protects mRNA from degradation by exoribonucleases but also interacts specifically with several proteins and enzymes to ensure mRNA turnover and processing, like splicing, export from the nucleus to the cytoplasm, and initiation of translation. The absence of a 5' cap leads to a strong immune response, and the methylation status contributes to distinguishing self from non-self RNA.Non-natural modifications of the 5' cap provide an avenue to label mRNAs and make them accessible to analyses, which is important to study their cellular localization, trafficking, and binding partners. They bear potential to engineer mRNAs, e.g., more stable or immunogenic mRNAs that are still translated, by impacting select interactions in a distinct manner. The modification of the 5' cap itself is powerful as it can be applied to make long mRNAs (∼1000 nt, not directly accessible by solid-phase synthesis) by in vitro transcription.This Account describes our contribution to the field of chemo-enzymatic modification of mRNA at the 5' cap. Our approach relies on RNA methyltransferases (MTases) with promiscuous activity on analogues of their natural cosubstrate S-adenosyl-L-methionine (AdoMet). We will describe how RNA MTases in combination with non-natural cosubstrates provide access to site-specific modification of different positions of the 5' cap, namely, the N2 and N7 position of guanosine and the N6 position of adenosine as the transcription start nucleotide (TSN) and exemplify strategies to make long mRNAs with modified 5' caps.We will compare the chemical and enzymatic synthesis of the AdoMet analogues used for this purpose. We could overcome previous limitations in methionine adenosyltransferase (MAT) substrate scope by engineering variants (termed PC-MATs) with the ability to convert methionine analogues with benzylic and photocaging groups at the sulfonium ion.The final part of this Account will highlight applications of the modified mRNAs. Like in many chemo-enzymatic approaches, a versatile strategy is to install small functional groups enzymatically and use them as handles in subsequent bioorthogonal reactions. We showed fluorescent labeling of mRNAs via different types of click chemistry in vitro and in cells. In a second line of applications, we used the handles to make mRNAs amenable for analyses, most notably next-generation sequencing. In the case of extremely promiscuous enzymes, the direct installation of photo-cross-linking groups was successful also and provided a way to covalently bind protein-interaction partners. Finally, the non-natural modifications of mRNAs can also modulate the properties of mRNAs. Propargylation of Am as the transcription start nucleotide at its N6 position maintained the translation of mRNAs but increased their immunogenicity. The installation of photocaging groups provides a way to revert these effects and control interactions by light.

COVID-19 and One-Carbon Metabolism.

Dysregulation of one-carbon metabolism affects a wide range of biological processes and is associated with a number of diseases, including cardiovascular disease, dementia, neural tube defects, and cancer. Accumulating evidence suggests that one-carbon metabolism plays an important role in COVID-19. The symptoms of long COVID-19 are similar to those presented by subjects suffering from vitamin B12 deficiency (pernicious anemia). The metabolism of a cell infected by the SARS-CoV-2 virus is reshaped to fulfill the need for massive viral RNA synthesis, which requires de novo purine biosynthesis involving folate and one-carbon metabolism. Many aspects of host sulfur amino acid metabolism, particularly glutathione metabolism underlying antioxidant defenses, are also taken over by the SARS-CoV-2 virus. The purpose of this review is to summarize recent findings related to one-carbon metabolism and sulfur metabolites in COVID-19 and discuss how they inform strategies to combat the disease.

Discrimination of Methionine Sulfoxide and Sulfone by Human Neutrophil Elastase.

Human neutrophil elastase (HNE) is a uniquely destructive serine protease with the ability to unleash a wave of proteolytic activity by destroying the inhibitors of other proteases. Although this phenomenon forms an important part of the innate immune response to invading pathogens, it is responsible for the collateral host tissue damage observed in chronic conditions such as chronic obstructive pulmonary disease (COPD), and in more acute disorders such as the lung injuries associated with COVID-19 infection. Previously, a combinatorially selected activity-based probe revealed an unexpected substrate preference for oxidised methionine, which suggests a link to oxidative pathogen clearance by neutrophils. Here we use oxidised model substrates and inhibitors to confirm this observation and to show that neutrophil elastase is specifically selective for the di-oxygenated methionine sulfone rather than the mono-oxygenated methionine sulfoxide. We also posit a critical role for ordered solvent in the mechanism of HNE discrimination between the two oxidised forms methionine residue. Preference for the sulfone form of oxidised methionine is especially significant. While both host and pathogens have the ability to reduce methionine sulfoxide back to methionine, a biological pathway to reduce methionine sulfone is not known. Taken together, these data suggest that the oxidative activity of neutrophils may create rapidly cleaved elastase "super substrates" that directly damage tissue, while initiating a cycle of neutrophil oxidation that increases elastase tissue damage and further neutrophil recruitment.

Dual-Mode Gradient HPLC and TLC Densitometry Methods for the Simultaneous Determination of Paracetamol and Methionine in the Presence of Paracetamol Impurities.

BACKGROUND: Paracetamol (PC) is one of the most widely used analgesic and antipyretic drugs and has recently been integrated into the supportive therapy of COVID-19. Pharmaceuticals containing methionine (MT) with PC may contribute to avoid hepatotoxicity and eventual PC overdose-dependent death. OBJECTIVE: The current work purposes to develop and validate two chromatographic methods for the simultaneous determination of MT and PC in the presence of two PC impurities (4-nitrophenol [NP] and 4-aminophenol [AP]). METHOD: Two chromatographic methods were established and validated according to the International Conference on Harmonization guidelines. The first one was an RP-HPLC/UV method based on applying a "dual-mode" gradient elution. The separation was realized via varying both the composition of the ternary mobile phase (acetonitrile-methanol-water) and its flow rate. This strategy enabled a relatively rapid analysis with a satisfactory resolution, although the investigated compounds exhibit a significant difference in lipophilicity. The second one relied on TLC-densitometry, where the optimum separation was realized using a quaternary mobile phase system composed of butanol-dioxane-toluene-methanol (8:2.5:3.5:0.3, by volume). Both methods were monitored at 220 nm. RESULTS: The developed methods were proven to be robust, accurate, specific, and appropriate for the routine analysis of PC in its pure form or in pharmaceutical formulations with MT in quality control laboratories. CONCLUSIONS: The corresponding methods are suitable to determine MT and PC in the presence of PC impurities. HIGHLIGHTS: The study achieves the analysis of MT and PC in the presence of PC impurities via the application of HPLC and TLC-densitometry methods.

Biomedical applications of methionine-based systems.

Methionine (Met), an essential amino acid in the human body, possesses versatile features based on its chemical modification, cell metabolism and metabolic derivatives. Benefitting from its multifunctional properties, Met holds immense potential for biomedical applications. In this review, we systematically summarize the recent progress in Met-based strategies for biomedical applications. First, given the unique structural characteristics of Met, two chemical modification methods are briefly introduced. Subsequently, due to the disordered metabolic state of tumor cells, applications of Met in cancer treatment and diagnosis are summarized in detail. Furthermore, the efficacy of S-adenosylmethionine (SAM), as the most important metabolic derivative of Met, for treating liver diseases is mentioned. Finally, we analyze the current challenges and development trends of Met in the biomedical field, and suggest that Met-restriction therapy might be a promising approach to treat COVID-19.

COVID-19 infection alters kynurenine and fatty acid metabolism, correlating with IL-6 levels and renal status.

BACKGROUNDReprogramming of host metabolism supports viral pathogenesis by fueling viral proliferation, by providing, for example, free amino acids and fatty acids as building blocks.METHODSTo investigate metabolic effects of SARS-CoV-2 infection, we evaluated serum metabolites of patients with COVID-19 (n = 33; diagnosed by nucleic acid testing), as compared with COVID-19-negative controls (n = 16).RESULTSTargeted and untargeted metabolomics analyses identified altered tryptophan metabolism into the kynurenine pathway, which regulates inflammation and immunity. Indeed, these changes in tryptophan metabolism correlated with interleukin-6 (IL-6) levels. Widespread dysregulation of nitrogen metabolism was also seen in infected patients, with altered levels of most amino acids, along with increased markers of oxidant stress (e.g., methionine sulfoxide, cystine), proteolysis, and renal dysfunction (e.g., creatine, creatinine, polyamines). Increased circulating levels of glucose and free fatty acids were also observed, consistent with altered carbon homeostasis. Interestingly, metabolite levels in these pathways correlated with clinical laboratory markers of inflammation (i.e., IL-6 and C-reactive protein) and renal function (i.e., blood urea nitrogen).CONCLUSIONIn conclusion, this initial observational study identified amino acid and fatty acid metabolism as correlates of COVID-19, providing mechanistic insights, potential markers of clinical severity, and potential therapeutic targets.FUNDINGBoettcher Foundation Webb-Waring Biomedical Research Award; National Institute of General and Medical Sciences, NIH; and National Heart, Lung, and Blood Institute, NIH.

Oral Methioninase for Covid-19 Methionine-restriction Therapy.

The Covid-19 pandemic is a world-wide crisis without an effective therapy. While most approaches to therapy are using repurposed drugs that were developed for other diseases, it is thought that targeting the biology of the SARS-CoV-2 virus, which causes Covid-19, can result in an effective therapeutic treatment. The coronavirus RNA cap structure is methylated by two viral methyltransferases that transfer methyl groups from S-adenosylmethionine (SAM). The proper methylation of the virus depends on the level of methionine in the host to form SAM. Herein, we propose to restrict methionine availability by treating the patient with oral recombinant methioninase, aiming to treat Covid-19. By restricting methionine we not only interdict viral replication, which depends on the viral RNA cap methyaltion, but also inhibit the proliferation of the infected cells, which have an increased requirement for methionine. Most importantly, the virally-induced T-cell- and macrophage-mediated cytokine storm, which seems to be a significant cause for Covid-19 deaths, can also be inhibited by restricting methionine, since T-cell and macrophrage activation greatly increases the methionine requirement for these cells. The evidence reviewed here suggests that oral recombinant methioninase could be a promising treatment for coronavirus patients.