Kyasanur Forest disease virus NS3 helicase: Insights into structure, activity, and inhibitors.

Kyasanur Forest disease virus (KFDV), a tick-borne flavivirus prevalent in India, presents a serious threat to human health. KFDV NS3 helicase (NS3hel) is considered a potential drug target due to its involvement in the viral replication complex. Here, we resolved the crystal structures of KFDV NS3hel apo and its complex with three phosphate molecules, which indicates a conformational switch during ATP hydrolysis. Our data revealed that KFDV NS3hel has a higher binding affinity for dsRNA, and its intrinsic ATPase activity was enhanced by dsRNA while being inhibited by DNA. Through mutagenesis analysis, several residues within motifs I, Ia, III, V, and VI were identified to be crucial for NS3hel ATPase activity. Notably, the M419A mutation drastically reduced NS3hel ATPase activity. We propose that the methionine-aromatic interaction between residues M419 and W294, located on the surface of the RNA-binding channel, could be a target for the design of efficient inhibitor probes. Moreover, epigallocatechin gallate (EGCG), a tea-derived polyphenol, strongly inhibited NS3hel ATPase activity with an IC50 value of 0.8 µM. Our computational docking data show that EGCG binds at the predicted druggable hotspots of NS3hel. Overall, these findings contribute to the development and design of more effective and specific inhibitors.

Structural mechanism of dsDNA recognition by the hMNDA HIN domain: New insights into the DNA-binding model of a PYHIN protein.

The hematopoietic interferon-inducible nuclear (HIN) domain of the PYHIN family of proteins recognizes double-stranded DNA (dsDNA) through different dsDNA-binding modes. These modes apparently confer different roles upon these proteins in the regulation of innate immune responses, gene transcription, and apoptosis. Myeloid cell nuclear differentiation antigen (MNDA), a member of the human PYHIN family, binds DNA and regulates gene transcription in monocytes. However, the mechanism of DNA recognition and DNA-binding modes of human MNDA (hMNDA) remain unclear. Here, we determined the crystal structure of the hMNDA-HIN domain in complex with dsDNA at 2.4 Å resolution, and reveal that hMNDA-HIN binds to dsDNA in a sequence-independent manner. Structure and mutation studies indicated that hMNDA-HIN binds to dsDNA through a unique mode, involving two dsDNA-binding interfaces. Interface I exhibits an AIM2-like dsDNA-binding mode, and interface II has a previously unreported mode of dsDNA-binding. These results provide new insights into the DNA-binding modes of this PYHIN protein.

PHLDA1 knockdown alleviates mitochondrial dysfunction and endoplasmic reticulum stress-induced neuronal apoptosis via activating PPARγ in cerebral ischemia-reperfusion injury.

Mitochondrial dysfunction and endoplasmic reticulum (ER) stress occur in ischemic stroke. The disruption of these two organelles can directly lead to cell death through various signaling pathways. Thus, investigation of the associated molecular mechanisms in cerebral ischemia is a prerequisite for stroke treatment. Pleckstrin homology-like domain family A member 1 (PHLDA1) is a multifunctional protein that can modulate mitochondrial function and ER stress in cardiomyocyte and cancer cells. This work studied the role of PHLDA1 in cerebral ischemic/reperfusion (I/R) injury and explored the underlying mechanisms associated with mitochondrial functions and ER stress. Middle cerebral artery occlusion/reperfusion (MCAO/R)-treated mice and oxygen-glucose deprivation/reoxygenation (OGD/R)-stimulated neurons were used as I/R models in vivo and in vitro, respectively. PHLDA1 was upregulated in ischemic penumbra of MCAO/R-induced mice and OGD/R-exposed neurons. In vitro, PHLDA1 knockdown protected neurons from OGD/R-induced apoptosis. In vivo, PHLDA1 silencing facilitated functional recovery and reduced cerebral infarct volume. Mechanistically, PHLDA1 knockdown promoted PPARγ nuclear translocation, which may mediate the effects on reversion of mitochondrial functions and alleviation of ER stress. In summary, PHLDA1 knockdown alleviates neuronal ischemic injuries in mice. PPARγ activation and mitochondrial dysfunction and endoplasmic reticulum stress attenuation are involved in the underlying mechanisms.

Toxicological mechanism of large amount of copper supplementation: Effects on endoplasmic reticulum stress and mitochondria-mediated apoptosis by Nrf2/HO-1 pathway-induced oxidative stress in the porcine myocardium.

Copper (Cu) is an essential micronutrient that is required by all living organisms. However, Cu can also be a potentially toxic metal if excessive dietary supplementation occurs. The current study aimed to investigate the mechanism of Cu toxicity in the cardiomyocytes of large mammal pigs. Here, we used pigs to explore Cu toxicity in the control group (10 mg/kg Cu) and treatment groups (125 mg/kg and 250 mg/kg Cu) for a period of 80 days. Consequently, we identified that large amount intake of Cu led to in oxidative damage, and activation of the nuclear factor erythroid 2-related factor 2 (Nrf2)/heme oxygenase-1 (HO-1)-mediated antioxidant pathway, indicating an imbalanced redox status in the myocardium. Furthermore, Cu exposure activated endoplasmic reticulum (ER) stress through upregulating levels of glucose-regulated protein 78 (GRP78), c-Jun N-terminal kinase (JNK), glucose-regulated protein 94 (GRP94), X-box binding protein 1 (XBP1), and C/EBP homologous protein (CHOP). Additionally, mitochondrial fission and fusion homeostasis was disrupted and the copy number of mitochondrial DNA (mtDNA) was reduced under Cu exposure. Furthermore, Cu exposure could induce apoptosis, evidenced by the increased terminal deoxynucleotidyl transferase biotin-d UTP nick end labeling (TUNEL)-positive staining, the upregulated expression levels of Cytoplasm-cytochrome C (Cytc), Bcl-2-associated X protein (Bax), and Cleaved-caspase3, and decreased expression level of B-cell lymphoma-2 (Bcl-2) and Mitochondrial-cytc. In summary, large amount of Cu could trigger Nrf2/HO-1 pathway-mediated oxidative stress, which promotes ER stress and mitochondrial damage pathways, causing apoptosis in cardiomyocytes.

Structure and mutation analysis of the hexameric P4 from Pseudomonas aeruginosa phage phiYY.

phiYY is a foremost member of Cystoviridae isolated from Pseudomonas aeruginosa. Its P4 protein with NTPase activity is a molecular motor for their genome packing during viral particle assembly. Previously studies on the P4 from four Pseudomonas phages phi6, phi8, phi12 and phi13 reveal that despite of belonging to the same protein family, they are unique in sequence, structure and biochemical properties. To better understand the structure and function of phiYY P4, four crystal structures of phiYY P4 in apo-form or combined with different ligands were solved at the resolution between 1.85 Å and 2.43 Å, which showed drastic conformation change of the H1 motif in ligand-bound forms compared with in apo-form, a four residue-mutation at the ligand binding pocket abolished its ATPase activity. Furthermore, the truncation mutation of the 50 residues at the C-terminal did not impair the hexamerization and ATP hydrolysis.

Structure of the NS5 methyltransferase from Zika virus and implications in inhibitor design.

Recent outbreak of flavivirus Zika virus (ZIKV) in America has urged the basic as well as translational studies of this important human pathogen. The nonstructural protein 5 (NS5) of the flavivirus has an N-terminal methyltransferase (MTase) domain that plays critical roles in viral RNA genome capping. The null mutant of NS5 MTase is lethal for virus. Therefore, NS5 is a potential drug target for the treatment of Zika virus infection. In this study, we determined crystal structures of the ZIKV MTase in complex with GTP and RNA cap analogue 7meGpppA. Structural analyses revealed highly conserved GTP/cap-binding pocket and S-adenosylmethionine (SAM)-binding pocket. Two conformations of the second base of the cap were identified, which suggests the flexibility of RNA conformation. In addition, the ligand-binding pockets identified a continuous region of hotspots suitable for drug design. Docking calculation shows that the Dengue virus inhibitor compound 10 may bind to the ZIKV MTase.