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56,413 results on '"⁎, N."'

10. Reconstitution of the SARS-CoV-2 ribonucleosome provides insights into genomic RNA packaging and regulation by phosphorylation

Author

Carlson, Christopher R, Adly, Armin N, Bi, Maxine, Howard, Conor J, Frost, Adam, Cheng, Yifan, and Morgan, David O

Subjects
Biological Sciences, Bioinformatics and Computational Biology, Prevention, Biodefense, Lung, Infectious Diseases, Vaccine Related, Genetics, Human Genome, 2.2 Factors relating to the physical environment, Underpinning research, Aetiology, 1.1 Normal biological development and functioning, Generic health relevance, Infection, Humans, SARS-CoV-2, Phosphorylation, RNA, Viral, COVID-19, Nucleocapsid Proteins, Ribonucleoproteins, Genomics, N protein, RNA binding protein, RNA virus, nucleocapsid, nucleosome, phosphorylation, plus-stranded RNA virus, Chemical Sciences, Medical and Health Sciences, Biochemistry & Molecular Biology, Biological sciences, Biomedical and clinical sciences, Chemical sciences
Abstract

The nucleocapsid (N) protein of severe acute respiratory syndrome coronavirus 2 is responsible for compaction of the ∼30-kb RNA genome in the ∼90-nm virion. Previous studies suggest that each virion contains 35 to 40 viral ribonucleoprotein (vRNP) complexes, or ribonucleosomes, arrayed along the genome. There is, however, little mechanistic understanding of the vRNP complex. Here, we show that N protein, when combined in vitro with short fragments of the viral genome, forms 15-nm particles similar to the vRNP structures observed within virions. These vRNPs depend on regions of N protein that promote protein-RNA and protein-protein interactions. Phosphorylation of N protein in its disordered serine/arginine region weakens these interactions to generate less compact vRNPs. We propose that unmodified N protein binds structurally diverse regions in genomic RNA to form compact vRNPs within the nucleocapsid, while phosphorylation alters vRNP structure to support other N protein functions in viral transcription.

Published
2022

11. The role of FXR and TGR5 in reversing and preventing progression of Western diet–induced hepatic steatosis, inflammation, and fibrosis in mice

Author

Wang, Xiaoxin X, Xie, Cen, Libby, Andrew E, Ranjit, Suman, Levi, Jonathan, Myakala, Komuraiah, Bhasin, Kanchan, Jones, Bryce A, Orlicky, David J, Takahashi, Shogo, Dvornikov, Alexander, Kleiner, David E, Hewitt, Stephen M, Adorini, Luciano, Kopp, Jeffrey B, Krausz, Kristopher W, Rosenberg, Avi, McManaman, James L, Robertson, Charles E, Ir, Diana, Frank, Daniel N, Luo, Yuhuan, Gonzalez, Frank J, Gratton, Enrico, and Levi, Moshe

Subjects
Nutrition, Chronic Liver Disease and Cirrhosis, Hepatitis, Prevention, Liver Disease, Obesity, Digestive Diseases, 2.1 Biological and endogenous factors, Aetiology, Metabolic and endocrine, Oral and gastrointestinal, Animals, Male, Mice, Bile Acids and Salts, Cholesterol, Diabetes Mellitus, Type 2, Diet, Western, Fatty Acids, Fibrosis, Inflammation, Mice, Inbred C57BL, Mice, Knockout, Non-alcoholic Fatty Liver Disease, Receptors, G-Protein-Coupled, FXR-TGR5, NASH, bile acid, fibrosis, inflammation, lipid metabolism, Chemical Sciences, Biological Sciences, Medical and Health Sciences, Biochemistry & Molecular Biology
Abstract

Nonalcoholic steatohepatitis (NASH) is the most common chronic liver disease in the US, partly due to the increasing incidence of metabolic syndrome, obesity, and type 2 diabetes. The roles of bile acids and their receptors, such as the nuclear receptor farnesoid X receptor (FXR) and the G protein-coupled receptor TGR5, on the development of NASH are not fully clear. C57BL/6J male mice fed a Western diet (WD) develop characteristics of NASH, allowing determination of the effects of FXR and TGR5 agonists on this disease. Here we show that the FXR-TGR5 dual agonist INT-767 prevents progression of WD-induced hepatic steatosis, inflammation, and fibrosis, as determined by histological and biochemical assays and novel label-free microscopy imaging techniques, including third harmonic generation, second harmonic generation, and fluorescence lifetime imaging microscopy. Furthermore, we show INT-767 decreases liver fatty acid synthesis and fatty acid and cholesterol uptake, as well as liver inflammation. INT-767 markedly changed bile acid composition in the liver and intestine, leading to notable decreases in the hydrophobicity index of bile acids, known to limit cholesterol and lipid absorption. In addition, INT-767 upregulated expression of liver p-AMPK, SIRT1, PGC-1α, and SIRT3, which are master regulators of mitochondrial function. Finally, we found INT-767 treatment reduced WD-induced dysbiosis of gut microbiota. Interestingly, the effects of INT-767 in attenuating NASH were absent in FXR-null mice, but still present in TGR5-null mice. Our findings support treatment and prevention protocols with the dual FXR-TGR5 agonist INT-767 arrest progression of WD-induced NASH in mice mediated by FXR-dependent, TGR5-independent mechanisms.

Published
2022

18. Mild membrane depolarization in neurons induces immediate early gene transcription and acutely subdues responses to a successive stimulus

Author

Rienecker, Kira DA, Poston, Robert G, Segales, Joshua S, Finholm, Isabelle W, Sono, Morgan H, Munteanu, Sorina J, Ghaninejad-Esfahani, Mina, Rejepova, Ayna, Tejeda-Garibay, Susana, Wickman, Kevin, de Velasco, Ezequiel Marron Fernandez, Thayer, Stanley A, and Saha, Ramendra N

Subjects
Biomedical and Clinical Sciences, Neurosciences, Genetics, Aetiology, 1.1 Normal biological development and functioning, 2.1 Biological and endogenous factors, Underpinning research, Neurological, Action Potentials, Animals, Bicuculline, Calcineurin, Calcium, GABA-A Receptor Antagonists, Genes, Immediate-Early, Mitogen-Activated Protein Kinases, Neurons, Potassium, Rats, Receptors, N-Methyl-D-Aspartate, Transcription, Genetic, immediate early genes, intrinsic excitability, membrane depolarization, neuronal activity, transcription, Chemical Sciences, Biological Sciences, Medical and Health Sciences, Biochemistry & Molecular Biology, Biological sciences, Biomedical and clinical sciences, Chemical sciences
Abstract

Immediate early genes (IEGs) are transcribed in response to neuronal activity from sensory stimulation during multiple adaptive processes in the brain. The transcriptional profile of IEGs is indicative of the duration of neuronal activity, but its sensitivity to the strength of depolarization remains unknown. Also unknown is whether activity history of graded potential changes influence future neuronal activity. In this work with dissociated rat cortical neurons, we found that mild depolarization-mediated by elevated extracellular potassium (K+)-induces a wide array of rapid IEGs and transiently depresses transcriptional and signaling responses to a successive stimulus. This latter effect was independent of de novo transcription, translation, and signaling via calcineurin or mitogen-activated protein kinase. Furthermore, as measured by multiple electrode arrays and calcium imaging, mild depolarization acutely subdues subsequent spontaneous and bicuculline-evoked activity via calcium- and N-methyl-d-aspartate receptor-dependent mechanisms. Collectively, this work suggests that a recent history of graded potential changes acutely depress neuronal intrinsic properties and subsequent responses. Such effects may have several potential downstream implications, including reducing signal-to-noise ratio during synaptic plasticity processes.

Published
2022

30. Regulatory subunit NEMO promotes polyubiquitin-dependent induction of NF-κB through a targetable second interaction with upstream activator IKK2

Author

Ko, Myung Soo, Cohen, Samantha N, Polley, Smarajit, Mahata, Sushil K, Biswas, Tapan, Huxford, Tom, and Ghosh, Gourisankar

Subjects
Biochemistry and Cell Biology, Biological Sciences, Humans, I-kappa B Kinase, NF-kappa B, Polyubiquitin, Protein Binding, NF-κB, enzyme inactivation, peptide interaction, polyubiquitin chain, protein kinase, Chemical Sciences, Medical and Health Sciences, Biochemistry & Molecular Biology, Biological sciences, Biomedical and clinical sciences, Chemical sciences
Abstract

Canonical NF-κB signaling through the inhibitor of κB kinase (IKK) complex requires induction of IKK2/IKKβ subunit catalytic activity via specific phosphorylation within its activation loop. This process is known to be dependent upon the accessory ubiquitin (Ub)-binding subunit NF-κB essential modulator (NEMO)/IKKγ as well as poly-Ub chains. However, the mechanism through which poly-Ub binding serves to promote IKK catalytic activity is unclear. Here, we show that binding of NEMO/IKKγ to linear poly-Ub promotes a second interaction between NEMO/IKKγ and IKK2/IKKβ, distinct from the well-characterized interaction of the NEMO/IKKγ N terminus to the "NEMO-binding domain" at the C terminus of IKK2/IKKβ. We mapped the location of this second interaction to a stretch of roughly six amino acids immediately N-terminal to the zinc finger domain in human NEMO/IKKγ. We also showed that amino acid residues within this region of NEMO/IKKγ are necessary for binding to IKK2/IKKβ through this secondary interaction in vitro and for full activation of IKK2/IKKβ in cultured cells. Furthermore, we identified a docking site for this segment of NEMO/IKKγ on IKK2/IKKβ within its scaffold-dimerization domain proximal to the kinase domain-Ub-like domain. Finally, we showed that a peptide derived from this region of NEMO/IKKγ is capable of interfering specifically with canonical NF-κB signaling in transfected cells. These in vitro biochemical and cell culture-based experiments suggest that, as a consequence of its association with linear poly-Ub, NEMO/IKKγ plays a direct role in priming IKK2/IKKβ for phosphorylation and that this process can be inhibited to specifically disrupt canonical NF-κB signaling.

Published
2022

48. Iron loading induces cholesterol synthesis and sensitizes endothelial cells to TNFα-mediated apoptosis

Author

Fisher, Allison L, Srole, Daniel N, Palaskas, Nicolaos J, Meriwether, David, Reddy, Srinivasa T, Ganz, Tomas, and Nemeth, Elizabeta

Subjects
Biochemistry and Cell Biology, Biomedical and Clinical Sciences, Biological Sciences, 2.1 Biological and endogenous factors, Aetiology, Cardiovascular, Apoptosis, Cholesterol, Ferric Compounds, Human Umbilical Vein Endothelial Cells, Humans, Iron, Iron Overload, Quaternary Ammonium Compounds, Tumor Necrosis Factor-alpha, SREBP, TNFR1, apoptosis, caspase, cholesterol metabolism, iron metabolism, lipid raft, tumor necrosis factor, Chemical Sciences, Medical and Health Sciences, Biochemistry & Molecular Biology, Biological sciences, Biomedical and clinical sciences, Chemical sciences
Abstract

In plasma, iron is normally bound to transferrin, the principal protein in blood responsible for binding and transporting iron throughout the body. However, in conditions of iron overload when the iron-binding capacity of transferrin is exceeded, non-transferrin-bound iron (NTBI) appears in plasma. NTBI is taken up by hepatocytes and other parenchymal cells via NTBI transporters and can cause cellular damage by promoting the generation of reactive oxygen species. However, how NTBI affects endothelial cells, the most proximal cell type exposed to circulating NTBI, has not been explored. We modeled in vitro the effects of systemic iron overload on endothelial cells by treating primary human umbilical vein endothelial cells (HUVECs) with NTBI (ferric ammonium citrate [FAC]). We showed by RNA-Seq that iron loading alters lipid homeostasis in HUVECs by inducing sterol regulatory element-binding protein 2-mediated cholesterol biosynthesis. We also determined that FAC increased the susceptibility of HUVECs to apoptosis induced by tumor necrosis factor-α (TNFα). Moreover, we showed that cholesterol biosynthesis contributes to iron-potentiated apoptosis. Treating HUVECs with a cholesterol chelator hydroxypropyl-β-cyclodextrin demonstrated that depletion of cholesterol was sufficient to rescue HUVECs from TNFα-induced apoptosis, even in the presence of FAC. Finally, we showed that FAC or cholesterol treatment modulated the TNFα pathway by inducing novel proteolytic processing of TNFR1 to a short isoform that localizes to lipid rafts. Our study raises the possibility that iron-mediated toxicity in human iron overload disorders is at least in part dependent on alterations in cholesterol metabolism in endothelial cells, increasing their susceptibility to apoptosis.

Published
2021

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