Putative Secondary Structure at 5’UTR as a Potential Antiviral Target against SARS-CoV-2

SARS-CoV-2 is an enveloped positive-sense single-stranded RNA coronavirus that causes COVID-19, of which the current outbreak has resulted in a high number of cases and fatalities throughout the world, even vaccine doses are being administered. The aim of this work was to scan the SARS-CoV-2 genome in search for therapeutic targets. We found a sequence in the 5’UTR (NC 045512:74-130), consisting of a typical heptamer next to a structured region that may cause ribosomal frameshifting. The potential biological value of this region is relevant through its low similarity with other viruses, including coronaviruses related to SARS-CoV, and its high sequence conservation within multiple SARS-CoV-2 isolates. We have predicted the secondary structure of the region by means of different bioinformatic tools. We have suggested a most probable secondary structure to proceed with a 3D reconstruction of the structured segment. Finally, we carried out virtual docking on the 3D structure to look for a binding site and then for drug ligands from a database of lead compounds. Several molecules that could be probably administered as oral drugs show promising binding affinity within the structured region, and so it could be possible interfere its potential regulatory role.

Insight into the Binding and Hydrolytic Preferences of hNudt16 Based on Nucleotide Diphosphate Substrates

Nudt16 is a member of the NUDIX family of hydrolases that show specificity towards substrates consisting of a nucleoside diphosphate linked to another moiety X. Several substrates for hNudt16 and various possible biological functions have been reported. However, some of these reports contradict each other and studies comparing the substrate specificity of the hNudt16 protein are limited. Therefore, we quantitatively compared the affinity of hNudt16 towards a set of previously published substrates, as well as identified novel potential substrates. Here, we show that hNudt16 has the highest affinity towards IDP and GppG, with Kd below 100 nM. Other tested ligands exhibited a weaker affinity of several orders of magnitude. Among the investigated compounds, only IDP, GppG, m7GppG, AppA, dpCoA, and NADH were hydrolyzed by hNudt16 with a strong substrate preference for inosine or guanosine containing compounds. A new identified substrate for hNudt16, GppG, which binds the enzyme with an affinity comparable to that of IDP, suggests another potential regulatory role of this protein. Molecular docking of hNudt16-ligand binding inside the hNudt16 pocket revealed two binding modes for representative substrates. Nucleobase stabilization by Π stacking interactions with His24 has been associated with strong binding of hNudt16 substrates.

Redox status of cysteines does not alter functional properties of human dUTPase but the Y54C mutation involved in monogenic diabetes decreases protein stability

Recently it was proposed that the redox status of cysteines acts as a redox switch to regulate both the oligomeric status and the activity of human dUTPase. In a separate report, a human dUTPase point mutation, resulting in a tyrosine to cysteine substitution (Y54C) was identified as the monogenic cause of a rare syndrome associated with diabetes and bone marrow failure. These issues prompt a critical investigation about the potential regulatory role of cysteines in the enzyme. Here we show on the one hand that independently of the redox status of wild-type cysteines, human dUTPase retains its characteristic trimeric assembly and its catalytic activity. On the other hand, the Y54C mutation did not compromise the substrate binding and the catalytic properties of the enzyme at room temperature. The thermal stability of the mutant protein was found to be decreased, which resulted in the loss of 67% of its activity after 90 min incubation at the physiological temperature in contrast to the wild-type enzyme. In addition, the presence or absence of reducing agents had no effect on hDUTY54C activity and stability, although it was confirmed that the introduced cysteine contains a solvent accessible thiol group.

Natural Killer T (NKT) Cells and Periodontitis: Potential Regulatory Role of NKT10 Cells

Natural killer T (NKT) cells constitute a unique subset of T lymphocytes characterized by specifically interacting with antigenic glycolipids conjugated to the CD1d receptor on antigen-presenting cells. Functionally, NKT cells are capable of performing either effector or suppressor immune responses, depending on their production of proinflammatory or anti-inflammatory cytokines, respectively. Effector NKT cells are subdivided into three subsets, termed NKT1, NKT2, and NKT17, based on the cytokines they produce and their similarity to the cytokine profile produced by Th1, Th2, and Th17 lymphocytes, respectively. Recently, a new subgroup of NKT cells termed NKT10 has been described, which cooperates and interacts with other immune cells to promote immunoregulatory responses. Although the tissue-specific functions of NKT cells have not been fully elucidated, their activity has been associated with the pathogenesis of different inflammatory diseases with immunopathogenic similarities to periodontitis, including osteolytic pathologies such as rheumatoid arthritis and osteoporosis. In the present review, we revise and discuss the pathogenic characteristics of NKT cells in these diseases and their role in the pathogenesis of periodontitis; particularly, we analyze the potential regulatory role of the IL-10-producing NKT10 cells.

PARylation prevents the proteasomal degradation of topoisomerase I DNA-protein crosslinks and induces their deubiquitylation

Poly(ADP)-ribosylation (PARylation) regulates chromatin structure and recruits DNA repair proteins. Using single-molecule fluorescence microscopy to track topoisomerase I (TOP1) in live cells, we found that sustained PARylation blocked the repair of TOP1 DNA-protein crosslinks (TOP1-DPCs) in a similar fashion as inhibition of the ubiquitin-proteasome system (UPS). PARylation of TOP1-DPC was readily revealed by inhibiting poly(ADP-ribose) glycohydrolase (PARG), indicating the otherwise transient and reversible PARylation of the DPCs. As the UPS is a key repair mechanism for TOP1-DPCs, we investigated the impact of TOP1-DPC PARylation on the proteasome and found that the proteasome is unable to associate with and digest PARylated TOP1-DPCs. In addition, PARylation recruits the deubiquitylating enzyme USP7 to reverse the ubiquitylation of PARylated TOP1-DPCs. Our work identifies PARG as repair factor for TOP1-DPCs by enabling the proteasomal digestion of TOP1-DPCs. It also suggests the potential regulatory role of PARylation for the repair of a broad range of DPCs.

Description of a Novel Fameshifting Site in the 5'UTR of SARS-CoV-2 as a Potential Drug Target

SARS-CoV-2 is an enveloped positive-sense single-stranded RNA coronavirus that causes COVID-19 whose present outbreak has cost a high number of casualties throughout the world. The aim of this work was to scan the SARS-CoV-2 genome in search for new therapeutic targets. We found a sequence in the 5'UTR (NC 045512:74-130), consisting of a typical heptamer next to a structured region that may cause frameshifting. The potential biological value of this region is shown by its similarity with other coronaviruses related with SARS-CoV and its sequence conservation within isolates from SARS-CoV-2. We have predicted the secondary structure of the region by means of different bioinformatic tools. We have chosen a probable secondary structure to proceed with a 3D reconstruction of the structured segment. We carried out virtual docking on the 3D structure to look for a binding site and then for drug ligands from a database of lead compounds. Several molecules that would probably administered as oral drugs show promising binding affinity within the structured region and so it would be possible interfere the potential regulatory role of our sequence of interest.

Nuclear factor-kappaB regulates the transcription of NADPH oxidase 1 in human alveolar epithelial cells

Objective Acute lung injury (ALI) is characterized by inflammation and oxidative stress. Nuclear factor-kappaB (NF-κB) mediates the expression of various inflammation-related genes, including the NADPH oxidase family. This study aimed to identify the potential regulatory role of NF-κB on NADPH oxidases in tumor necrosis factor-α (TNF-α)-induced oxidative stress in human alveolar epithelial cells. Methods A549 cells were treated with TNF-α for 24 h to establish ALI cell models. RT-PCR, western blot, assessment of oxidative stress, Alibaba 2.1 online analysis, electrophoretic mobility shift assays and luciferase reporter analysis were employed to identify the potential regulatory role of NF-κB on NADPH oxidases in TNF-α-induced oxidative stress in human alveolar epithelial cells. Results The expression of NF-κB/p65 was notably upregulated in TNF-α-stimulated A549 cells. NF-κB knockdown by siRNA significantly inhibited the TNF-α-induced oxidative stress. Moreover, NF-κB/p65 siRNA could inhibit the activation of NOX1, NOX2 and NOX4 mRNA and protein expression in TNF-α-stimulated A549 cells. The next study demonstrated that NF-κB activated the transcription of NOX1 by binding to the -261 to -252 bp (NOX1/κB2, TAAAAATCCC) region of NOX1 promoter in TNF-α-stimulated A549 cells. Conclusion Our data demonstrated that NF-κB can aggravate TNF-α-induced ALI by regulating the oxidative stress response and the expression of NOX1, NOX2 and NOX4. Moreover, NF-κB could promote the NOX1 transcriptional activity via binding its promoter in TNF-α-stimulated A549 cells.