Efficient De Novo Biosynthesis of Heme by Membrane Engineering in Escherichia coli.

Heme is of great significance in food nutrition and food coloring, and the successful launch of artificial meat has greatly improved the application of heme in meat products. The precursor of heme, 5-aminolevulinic acid (ALA), has a wide range of applications in the agricultural and medical fields, including in the treatment of corona virus disease 2019 (COVID-19). In this study, E. coli recombinants capable of heme production were developed by metabolic engineering and membrane engineering. Firstly, by optimizing the key genes of the heme synthesis pathway and the screening of hosts and plasmids, the recombinant strain EJM-pCD-AL produced 4.34 ± 0.02 mg/L heme. Then, the transport genes of heme precursors CysG, hemX and CyoE were knocked out, and the extracellular transport pathways of heme Dpp and Ccm were strengthened, obtaining the strain EJM-ΔCyoE-pCD-AL that produced 9.43 ± 0.03 mg/L heme. Finally, fed-batch fermentation was performed in a 3-L fermenter and reached 28.20 ± 0.77 mg/L heme and 303 ± 1.21 mg/L ALA. This study indicates that E. coli recombinant strains show a promising future in the field of heme and ALA production.

[A new approach to combat the sepsis including COVID-19 by accelerating detoxification of hemolysis-related DAMPs].

Sepsis is one of the leading cause of death worldwide. Recently, several studies suggested that free-hemoglobin and heme derived from hemolysis are important factors which may be associated with severity of septic patients including COVID-19. In other words, hemolysis-derived products enhance the inflammatory responses as damage-associated molecular patterns (DAMPs) in both intravascular and extravascular space. In addition, hemoglobin has vasoconstrictive activity by depleting nitric oxide, whereas heme or Fe2+ produce reactive oxygen species (ROS) through Fenton reaction leading to tissue injury. At present, we have no therapeutic options against sepsis-related hemolysis in clinical settings, however, there might be two therapeutic strategies in this regard. One is supplemental therapy of depleted scavenging proteins such as haptoglobin and hemopexin, the other is activation of the internal scavenging system including macrophage-CD163 pathway. These novel targets against sepsis are also critical for the next pandemic. In this review, we summarize the current issues regarding sepsis-related hemolysis including COVID-19, as well as for future perspectives.

The Mechanism-Based Inactivation of CYP3A4 by Ritonavir: What Mechanism?

Ritonavir is the most potent cytochrome P450 (CYP) 3A4 inhibitor in clinical use and is often applied as a booster for drugs with low oral bioavailability due to CYP3A4-mediated biotransformation, as in the treatment of HIV (e.g., lopinavir/ritonavir) and more recently COVID-19 (Paxlovid or nirmatrelvir/ritonavir). Despite its clinical importance, the exact mechanism of ritonavir-mediated CYP3A4 inactivation is still not fully understood. Nonetheless, ritonavir is clearly a potent mechanism-based inactivator, which irreversibly blocks CYP3A4. Here, we discuss four fundamentally different mechanisms proposed for this irreversible inactivation/inhibition, namely the (I) formation of a metabolic-intermediate complex (MIC), tightly coordinating to the heme group; (II) strong ligation of unmodified ritonavir to the heme iron; (III) heme destruction; and (IV) covalent attachment of a reactive ritonavir intermediate to the CYP3A4 apoprotein. Ritonavir further appears to inactivate CYP3A4 and CYP3A5 with similar potency, which is important since ritonavir is applied in patients of all ethnicities. Although it is currently not possible to conclude what the primary mechanism of action in vivo is, it is unlikely that any of the proposed mechanisms are fundamentally wrong. We, therefore, propose that ritonavir markedly inactivates CYP3A through a mixed set of mechanisms. This functional redundancy may well contribute to its overall inhibitory efficacy.

SARS-CoV-2 Infects Red Blood Cell Progenitors and Dysregulates Hemoglobin and Iron Metabolism.

BACKGROUND: SARS-CoV-2 infection causes acute respiratory distress, which may progress to multiorgan failure and death. Severe COVID-19 disease is accompanied by reduced erythrocyte turnover, low hemoglobin levels along with increased total bilirubin and ferritin serum concentrations. Moreover, expansion of erythroid progenitors in peripheral blood together with hypoxia, anemia, and coagulopathies highly correlates with severity and mortality. We demonstrate that SARS-CoV-2 directly infects erythroid precursor cells, impairs hemoglobin homeostasis and aggravates COVID-19 disease. METHODS: Erythroid precursor cells derived from peripheral CD34+ blood stem cells of healthy donors were infected in vitro with SARS-CoV-2 alpha variant and differentiated into red blood cells (RBCs). Hemoglobin and iron metabolism in hospitalized COVID-19 patients and controls were analyzed in plasma-depleted whole blood samples. Raman trapping spectroscopy rapidly identified diseased cells. RESULTS: RBC precursors express ACE2 receptor and CD147 at day 5 of differentiation, which makes them susceptible to SARS-CoV-2 infection. qPCR analysis of differentiated RBCs revealed increased HAMP mRNA expression levels, encoding for hepcidin, which inhibits iron uptake. COVID-19 patients showed impaired hemoglobin biosynthesis, enhanced formation of zinc-protoporphyrine IX, heme-CO2, and CO-hemoglobin as well as degradation of Fe-heme. Moreover, significant iron dysmetablolism with high serum ferritin and low serum iron and transferrin levels occurred, explaining disturbances of oxygen-binding capacity in severely ill COVID-19 patients. CONCLUSIONS: Our data identify RBC precursors as a direct target of SARS-CoV-2 and suggest that SARS-CoV-2 induced dysregulation in hemoglobin- and iron-metabolism contributes to the severe systemic course of COVID-19. This opens the door for new diagnostic and therapeutic strategies.

SARS-CoV-2 can recruit a heme metabolite to evade antibody immunity.

The coronaviral spike is the dominant viral antigen and the target of neutralizing antibodies. We show that SARS-CoV-2 spike binds biliverdin and bilirubin, the tetrapyrrole products of heme metabolism, with nanomolar affinity. Using cryo-electron microscopy and x-ray crystallography, we mapped the tetrapyrrole interaction pocket to a deep cleft on the spike N-terminal domain (NTD). At physiological concentrations, biliverdin significantly dampened the reactivity of SARS-CoV-2 spike with immune sera and inhibited a subset of neutralizing antibodies. Access to the tetrapyrrole-sensitive epitope is gated by a flexible loop on the distal face of the NTD. Accompanied by profound conformational changes in the NTD, antibody binding requires relocation of the gating loop, which folds into the cleft vacated by the metabolite. Our results indicate that SARS-CoV-2 spike NTD harbors a dominant epitope, access to which can be controlled by an allosteric mechanism that is regulated through recruitment of a metabolite.

Potential role of free-radical processes in biomolecules damage during COVID-19 and ways of their regulation.

It has been shown that the development of coronavirus infection (COVID-19), especially in severe cases, is accompanied by hypoxia as a result of several pathological processes: alveolar blood supply disorders, hemolysis, COVID-associated coagulopathy. Under these conditions, the level of reactive oxygen species is increased and it is more likely that free-radical damage to biomolecules is caused by the process of free-radical fragmentation than oxidation. In contrast to the oxidation process, free-radical fragmentation reactions are more effectively inhibited by oxidizing agents than reducing agents. Therefore, the use of substances possessing both reducing and oxidizing properties, such as natural and synthetic quinones, bioflavonoids, curcuminoids, should reduce the probability of biomolecule destruction by oxidation as well as free-radical fragmentation processes.HighlightsCOVID-19 is accompanied by the iron release from the heme and «silent¼ hypoxiaROS initiate fragmentation reactions of biomolecules under conditions of hypoxiaBlocking of fragmentation process by oxidizers may lead to mitigation of COVID-19.

Heme oxygenase-1 modulation: A potential therapeutic target for COVID-19 and associated complications.

Coronavirus disease 2019 (COVID-19) caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) continues to infect hundred thousands of people every day worldwide. Since it is a novel virus, research continues to update the possible therapeutic targets when new evidence regarding COVID-19 are gathered. This article presents an evidence-based hypothesis that activating the heme oxygenase-1 (HO-1) pathway is a potential target for COVID-19. Interferons (IFNs) have broad-spectrum antiviral activity including against SARS-CoV-2. Induction of HO-1 and increase in the heme catabolism end-product confer antiviral activity. IFN activation results in inhibition of viral replication in various viral infections. COVID-19 induced inflammation as well as acute respiratory distress syndrome (ARDS), and coagulopathies are now known major causes of mortality. A protective role of HO-1 induction in inflammation, inflammation-induced coagulation, and ARDS has been reported. Based on an association of HO-1 promoter polymorphisms and disease severity, we propose an evaluation of the status of these polymorphisms in COVID-19 patients who become severely ill. If an association is established, it might be helpful in identifying patients at high risk. Hence, we hypothesize that HO-1 pathway activation could be a therapeutic strategy against COVID-19 and associated complications.

Desaturation and heme elevation during COVID-19 infection: A potential prognostic factor of heme oxygenase-1.

Increased heme levels, anemia, and desaturation occur during infection. We aimed to compare the levels of heme, heme oxygenase-1 (HO-1), ferritin, and bilirubin in coronavirus disease 2019 (COVID-19) patients at different saturation levels. Heme and HO-1 enzyme levels significantly increased in the low SpO2 group, but further studies are required.