Generation and Characterization of CYP2E1-Overexpressing HepG2 Cells to Study the Role of CYP2E1 in Hepatic Hypoxia-Reoxygenation Injury.

The mechanisms of hepatic ischemia/reperfusion (I/R) injury, which occurs during liver transplantation or surgery, are poorly understood. The purpose of the current study was to generate and characterize a HepG2 cell line with a stable overexpression of CYP2E1 to investigate the role of the enzyme in hypoxia/reperfusion (H/R) injury in an ex vivo setting. GFP-tagged CYP2E1 and control clones were developed, and their gene expression and protein levels of GFP and CYP2E1 were determined using RT-PCR and ELISA/Western blot analysis, respectively. Additionally, the CYP2E1 catalytic activity was determined by UPLC-MS/MS analysis of 6-hydroxychlorzoxazone formed from the chlorzoxazone substrate. The CYP2E1 and control clones were subjected to hypoxia (10 h) and reoxygenation (0.5 h), and cell death and reactive oxygen species (ROS) generation were quantitated using LDH and flow cytometry, respectively. Compared with the control clone, the selected CYP2E1 clone showed a 720-fold increase in CYP2E1 expression and a prominent band in the western blot analysis, which was associated with a 150-fold increase in CYP2E1 catalytic activity. The CYP2E1 clone produced 2.3-fold more ROS and 1.9-fold more cell death in the H/R model. It is concluded that the constitutive CYP2E1 in the liver may play a detrimental role in hepatic I/R injury.

Structural and functional characterization of NEMO cleavage by SARS-CoV-2 3CLpro.

In addition to its essential role in viral polyprotein processing, the SARS-CoV-2 3C-like protease (3CLpro) can cleave human immune signaling proteins, like NF-κB Essential Modulator (NEMO) and deregulate the host immune response. Here, in vitro assays show that SARS-CoV-2 3CLpro cleaves NEMO with fine-tuned efficiency. Analysis of the 2.50 Å resolution crystal structure of 3CLpro C145S bound to NEMO226-234 reveals subsites that tolerate a range of viral and host substrates through main chain hydrogen bonds while also enforcing specificity using side chain hydrogen bonds and hydrophobic contacts. Machine learning- and physics-based computational methods predict that variation in key binding residues of 3CLpro-NEMO helps explain the high fitness of SARS-CoV-2 in humans. We posit that cleavage of NEMO is an important piece of information to be accounted for, in the pathology of COVID-19.

Structural and functional characterization of NEMO cleavage by SARS-CoV-2 3CLpro.

In addition to its essential role in viral polyprotein processing, the SARS-CoV-2 3C-like (3CLpro) protease can cleave human immune signaling proteins, like NF-κB Essential Modulator (NEMO) and deregulate the host immune response. Here, in vitro assays show that SARS-CoV-2 3CLpro cleaves NEMO with fine-tuned efficiency. Analysis of the 2.14 Å resolution crystal structure of 3CLpro C145S bound to NEMO 226-235 reveals subsites that tolerate a range of viral and host substrates through main chain hydrogen bonds while also enforcing specificity using side chain hydrogen bonds and hydrophobic contacts. Machine learning- and physics-based computational methods predict that variation in key binding residues of 3CLpro- NEMO helps explain the high fitness of SARS-CoV-2 in humans. We posit that cleavage of NEMO is an important piece of information to be accounted for in the pathology of COVID-19.

Cyclic Peptides as Protein Kinase Inhibitors: Structure-Activity Relationship and Molecular Modeling.

Under-expression or overexpression of protein kinases has been shown to be associated with unregulated cell signal transduction in cancer cells. Therefore, there is major interest in designing protein kinase inhibitors as anticancer agents. We have previously reported [WR]5, a peptide containing alternative arginine (R) and tryptophan (W) residues as a non-competitive c-Src tyrosine kinase inhibitor. A number of larger cyclic peptides containing alternative hydrophobic and positively charged residues [WR]x (x = 6-9) and hybrid cyclic-linear peptides, [R6K]W6 and [R5K]W7, containing R and W residues were evaluated for their protein kinase inhibitory potency. Among all the peptides, cyclic peptide [WR]9 was found to be the most potent tyrosine kinase inhibitor. [WR]9 showed higher inhibitory activity (IC50 = 0.21 µM) than [WR]5, [WR]6, [WR]7, and [WR]8 with IC50 values of 0.81, 0.57, 0.35, and 0.33 µM, respectively, against c-Src kinase as determined by a radioactive assay using [γ-33P]ATP. Consistent with the result above, [WR]9 inhibited other protein kinases such as Abl kinase activity with an IC50 value of 0.35 µM, showing 2.2-fold higher inhibition than [WR]5 (IC50 = 0.79 µM). [WR]9 also inhibited PKCa kinase activity with an IC50 value of 2.86 µM, approximately threefold higher inhibition than [WR]5 (IC50 = 8.52 µM). A similar pattern was observed against Braf, c-Src, Cdk2/cyclin A1, and Lck. [WR]9 exhibited IC50 values of <0.25 µM against Akt1, Alk, and Btk. These data suggest that [WR]9 is consistently more potent than other cyclic peptides with a smaller ring size and hybrid cyclic-linear peptides [R6K]W6 and [R5K]W7 against selected protein kinases. Thus, the presence of R and W residues in the ring, ring size, and the number of amino acids in the structure of the cyclic peptide were found to be critical in protein kinase inhibitory potency. We identified three putative binding pockets through automated blind docking of cyclic peptides [WR](5-9). The most populated pocket is located between the SH2, SH3, and N-lobe domains on the opposite side of the ATP binding site. The second putative pocket is formed by the same domains and located on the ATP binding site side of the protein. Finally, a third pocket was identified between the SH2 and SH3 domains. These results are consistent with the non-competitive nature of the inhibition displayed by these molecules. Molecular dynamics simulations of the protein-peptide complexes indicate that the presence of either [WR]5 or [WR]9 affects the plasticity of the protein and in particular the volume of the ATP binding site pocket in different ways. These results suggest that the second pocket is most likely the site where these peptides bind and offer a plausible rationale for the increased affinity of [WR]9.

Linear ubiquitin chain-binding domains.

Ubiquitin modification (ubiquitination) of target proteins can vary with respect to chain lengths, linkage type, and chain forms, such as homologous, mixed, and branched ubiquitin chains. Thus, ubiquitination can generate multiple unique surfaces on a target protein substrate. Ubiquitin-binding domains (UBDs) recognize ubiquitinated substrates, by specifically binding to these unique surfaces, modulate the formation of cellular signaling complexes and regulate downstream signaling cascades. Among the eight different homotypic chain types, Met1-linked (also termed linear) chains are the only chains in which linkage occurs on a non-Lys residue of ubiquitin. Linear ubiquitin chains have been implicated in immune responses, cell death and autophagy, and several UBDs - specific for linear ubiquitin chains - have been identified. In this review, we describe the main principles of ubiquitin recognition by UBDs, focusing on linear ubiquitin chains and their roles in biology.

Specific recognition of linear ubiquitin chains by NEMO is important for NF-kappaB activation.

Activation of nuclear factor-kappaB (NF-kappaB), a key mediator of inducible transcription in immunity, requires binding of NF-kappaB essential modulator (NEMO) to ubiquitinated substrates. Here, we report that the UBAN (ubiquitin binding in ABIN and NEMO) motif of NEMO selectively binds linear (head-to-tail) ubiquitin chains. Crystal structures of the UBAN motif revealed a parallel coiled-coil dimer that formed a heterotetrameric complex with two linear diubiquitin molecules. The UBAN dimer contacted all four ubiquitin moieties, and the integrity of each binding site was required for efficient NF-kappaB activation. Binding occurred via a surface on the proximal ubiquitin moiety and the canonical Ile44 surface on the distal one, thereby providing specificity for linear chain recognition. Residues of NEMO involved in binding linear ubiquitin chains are required for NF-kappaB activation by TNF-alpha and other agonists, providing an explanation for the detrimental effect of NEMO mutations in patients suffering from X-linked ectodermal dysplasia and immunodeficiency.