AggreCount: an unbiased image analysis tool for identifying and quantifying cellular aggregates in a spatially defined manner [Methods and Resources]
Protein quality control is maintained by a number of integrated cellular pathways that monitor the folding and functionality of the cellular proteome. Defects in these pathways lead to the accumulation of misfolded or faulty proteins that may become insoluble and aggregate over time. Protein aggregates significantly contribute to the development of a number of human diseases such as amyotrophic lateral sclerosis, Huntington's disease, and Alzheimer's disease. In vitro, imaging-based, cellular studies have defined key biomolecular components that recognize and clear aggregates; however, no unifying method is available to qu...
Source: Journal of Biological Chemistry - December 18, 2020 Category: Chemistry Authors: Jacob Aaron Klickstein, Sirisha Mukkavalli, Malavika Raman Tags: Cell Biology Source Type: research
AMPK{beta}1 and AMPK{beta}2 define an isoform-specific gene signature in human pluripotent stem cells, differentially mediating cardiac lineage specification [Cell Biology]
AMP-activated protein kinase (AMPK) is a key regulator of energy metabolism that phosphorylates a wide range of proteins to maintain cellular homeostasis. AMPK consists of three subunits: α, β, and γ. AMPKα and β are encoded by two genes, the γ subunit by three genes, all of which are expressed in a tissue-specific manner. It is not fully understood, whether individual isoforms have different functions. Using RNA-Seq technology, we provide evidence that the loss of AMPKβ1 and AMPKβ2 lead to different gene expression profiles in human induced pluripotent stem cells (hiPSCs), indicating isoform-specific function. The...
Source: Journal of Biological Chemistry - December 18, 2020 Category: Chemistry Authors: Nicole Ziegler, Erik Bader, Alexey Epanchintsev, Daniel Margerie, Aimo Kannt, Dieter Schmoll Tags: Metabolism Source Type: research
The HRDC domain oppositely modulates the unwinding activity of E. coli RecQ helicase on duplex DNA and G-quadruplex [Enzymology]
In this study, we systematically characterized the roles of the HRDC domain in E. coli RecQ in various DNA transactions by single-molecule FRET. We found that RecQ repetitively unwinds the 3′-partial duplex and fork DNA with a moderate processivity and periodically patrols on the ssDNA in the 5′-partial duplex by translocation. The HRDC domain significantly suppresses RecQ activities in the above transactions. In sharp contrast, the HRDC domain is essential for the deep and long-time unfolding of the G4 DNA structure by RecQ. Based on the observations that the HRDC domain dynamically switches between RecA core- and ssD...
Source: Journal of Biological Chemistry - December 18, 2020 Category: Chemistry Authors: Fang-Yuan Teng, Ting-Ting Wang, Hai-Lei Guo, Ben-Ge Xin, Bo Sun, Shuo-Xing Dou, Xu-Guang Xi, Xi-Miao Hou Tags: Molecular Biophysics Source Type: research
Importance of endothelial Hey1 expression for thoracic great vessel development and its distal enhancer for Notch-dependent endothelial transcription [Gene Regulation]
In this study, we revealed that Hey1 in vascular endothelial cells, but not in smooth muscle cells, played essential roles for PAA development and great vessel morphogenesis in mouse embryos. Tek-Cre–mediated Hey1 deletion in endothelial cells affected endothelial tube formation and smooth muscle differentiation in embryonic fourth PAAs and resulted in interruption of the aortic arch and other great vessel malformations. Cell specificity and signal responsiveness of Hey1 expression were controlled through multiple cis-regulatory regions. We found two distal genomic regions that had enhancer activity in endothelial cells ...
Source: Journal of Biological Chemistry - December 18, 2020 Category: Chemistry Authors: Yusuke Watanabe, Daiki Seya, Dai Ihara, Shuhei Ishii, Taiki Uemoto, Atsushi Kubo, Yuji Arai, Yoshie Isomoto, Atsushi Nakano, Takaya Abe, Mayo Shigeta, Teruhisa Kawamura, Yoshihiko Saito, Toshihiko Ogura, Osamu Nakagawa Tags: Developmental Biology Source Type: research
NETosis occurs independently of neutrophil serine proteases [Enzymology]
Neutrophils are primary host innate immune cells defending against pathogens. One proposed mechanism by which neutrophils prevent the spread of pathogens is NETosis, the extrusion of cellular DNA resulting in neutrophil extracellular traps (NETs). The protease neutrophil elastase (NE) has been implicated in the formation of NETs through proteolysis of nuclear proteins leading to chromatin decondensation. In addition to NE, neutrophils contain three other serine proteases that could compensate if the activity of NE was neutralized. However, whether they do play such a role is unknown. Thus, we deployed recently described sp...
Source: Journal of Biological Chemistry - December 18, 2020 Category: Chemistry Authors: Paulina Kasperkiewicz, Anne Hempel, Tomasz Janiszewski, Sonia Kołt, Scott J. Snipas, Marcin Drag, Guy S. Salvesen Tags: Cell Biology Source Type: research
Bacterial iron detoxification at the molecular level [Protein Structure and Folding]
Iron is an essential micronutrient, and, in the case of bacteria, its availability is commonly a growth-limiting factor. However, correct functioning of cells requires that the labile pool of chelatable “free” iron be tightly regulated. Correct metalation of proteins requiring iron as a cofactor demands that such a readily accessible source of iron exist, but overaccumulation results in an oxidative burden that, if unchecked, would lead to cell death. The toxicity of iron stems from its potential to catalyze formation of reactive oxygen species that, in addition to causing damage to biological molecules, can also lead ...
Source: Journal of Biological Chemistry - December 18, 2020 Category: Chemistry Authors: Justin M. Bradley, Dimitry A. Svistunenko, Michael T. Wilson, Andrew M. Hemmings, Geoffrey R. Moore, Nick E. Le Brun Tags: JBC Reviews Source Type: research
Visualizing, quantifying, and manipulating mitochondrial DNA in vivo [Methods and Resources]
Mitochondrial DNA (mtDNA) encodes proteins and RNAs that support the functions of mitochondria and thereby numerous physiological processes. Mutations of mtDNA can cause mitochondrial diseases and are implicated in aging. The mtDNA within cells is organized into nucleoids within the mitochondrial matrix, but how mtDNA nucleoids are formed and regulated within cells remains incompletely resolved. Visualization of mtDNA within cells is a powerful means by which mechanistic insight can be gained. Manipulation of the amount and sequence of mtDNA within cells is important experimentally and for developing therapeutic interventi...
Source: Journal of Biological Chemistry - December 18, 2020 Category: Chemistry Authors: David L. Prole, Patrick F. Chinnery, Nick S. Jones Tags: JBC Reviews Source Type: research
Wildtype {sigma}1 receptor and the receptor agonist improve ALS-associated mutation-induced insolubility and toxicity [Neurobiology]
Genetic mutations related to ALS, a progressive neurological disease, have been discovered in the gene encoding σ-1 receptor (σ1R). We previously reported that σ1RE102Q elicits toxicity in cells. The σ1R forms oligomeric states that are regulated by ligands. Nevertheless, little is known about the effect of ALS-related mutations on oligomer formation. Here, we transfected NSC-34 cells, a motor neuronal cell line, and HEK293T cells with σ1R-mCherry (mCh), σ1RE102Q-mCh, or nontagged forms to investigate detergent solubility and subcellular distribution using immunocytochemistry and fluorescence recovery after photoblea...
Source: Journal of Biological Chemistry - December 18, 2020 Category: Chemistry Authors: Yasuharu Shinoda, Yudai Haga, Koichiro Akagawa, Kohji Fukunaga Tags: Cell Biology Source Type: research
Hepatocyte nuclear factor 1{beta} suppresses canonical Wnt signaling through transcriptional repression of lymphoid enhancer-binding factor 1 [Molecular Bases of Disease]
Hepatocyte nuclear factor-1β (HNF-1β) is a tissue-specific transcription factor that is required for normal kidney development and renal epithelial differentiation. Mutations of HNF-1β produce congenital kidney abnormalities and inherited renal tubulopathies. Here, we show that ablation of HNF-1β in mIMCD3 renal epithelial cells results in activation of β-catenin and increased expression of lymphoid enhancer–binding factor 1 (LEF1), a downstream effector in the canonical Wnt signaling pathway. Increased expression and nuclear localization of LEF1 are also observed in cystic kidneys from Hnf1b mutant mice. Expression...
Source: Journal of Biological Chemistry - December 18, 2020 Category: Chemistry Authors: Siu Chiu Chan, Sachin S. Hajarnis, Sophia M. Vrba, Vishal Patel, Peter Igarashi Tags: Gene Regulation Source Type: research
Post-translational control of the long and winding road to cholesterol [Lipids]
The synthesis of cholesterol requires more than 20 enzymes, many of which are intricately regulated. Post-translational control of these enzymes provides a rapid means for modifying flux through the pathway. So far, several enzymes have been shown to be rapidly degraded through the ubiquitin–proteasome pathway in response to cholesterol and other sterol intermediates. Additionally, several enzymes have their activity altered through phosphorylation mechanisms. Most work has focused on the two rate-limiting enzymes: 3-hydroxy-3-methylglutaryl CoA reductase and squalene monooxygenase. Here, we review current literature in ...
Source: Journal of Biological Chemistry - December 18, 2020 Category: Chemistry Authors: Laura J. Sharpe, Hudson W. Coates, Andrew J. Brown Tags: JBC Reviews Source Type: research
The role of uncoupling protein 2 in macrophages and its impact on obesity-induced adipose tissue inflammation and insulin resistance [Immunology]
The development of a chronic, low-grade inflammation originating from adipose tissue in obese subjects is widely recognized to induce insulin resistance, leading to the development of type 2 diabetes. The adipose tissue microenvironment drives specific metabolic reprogramming of adipose tissue macrophages, contributing to the induction of tissue inflammation. Uncoupling protein 2 (UCP2), a mitochondrial anion carrier, is thought to separately modulate inflammatory and metabolic processes in macrophages and is up-regulated in macrophages in the context of obesity and diabetes. Here, we investigate the role of UCP2 in macrop...
Source: Journal of Biological Chemistry - December 18, 2020 Category: Chemistry Authors: Xanthe A. M. H. van Dierendonck, Tiphaine Sancerni, Marie-Clotilde Alves-Guerra, Rinke Stienstra Tags: Metabolism Source Type: research
Evolving the naturally compromised chorismate mutase from Mycobacterium tuberculosis to top performance [Protein Structure and Folding]
Chorismate mutase (CM), an essential enzyme at the branch-point of the shikimate pathway, is required for the biosynthesis of phenylalanine and tyrosine in bacteria, archaea, plants, and fungi. MtCM, the CM from Mycobacterium tuberculosis, has less than 1% of the catalytic efficiency of a typical natural CM and requires complex formation with 3-deoxy-d-arabino-heptulosonate 7-phosphate synthase for high activity. To explore the full potential of MtCM for catalyzing its native reaction, we applied diverse iterative cycles of mutagenesis and selection, thereby raising kcat/Km 270-fold to 5 × 105 m−1s−1, which is even hi...
Source: Journal of Biological Chemistry - December 18, 2020 Category: Chemistry Authors: Jūrate˙ Fahrig–Kamarauskait≑, Kathrin Wurth–Roderer, Helen V. Thorbȷornsrud, Susanne Mailand, Ute Krengel, Peter Kast Tags: Enzymology Source Type: research
Fibrillar {alpha}-synuclein toxicity depends on functional lysosomes [Cell Biology]
Neurodegeneration in Parkinson's disease (PD) can be recapitulated in animals by administration of α-synuclein preformed fibrils (PFFs) into the brain. However, the mechanism by which these PFFs induce toxicity is unknown. Iron is implicated in PD pathophysiology, so we investigated whether α-synuclein PFFs induce ferroptosis, an iron-dependent cell death pathway. A range of ferroptosis inhibitors were added to a striatal neuron-derived cell line (STHdhQ7/7 cells), a dopaminergic neuron–derived cell line (SN4741 cells), and WT primary cortical neurons, all of which had been intoxicated with α-synuclein PFFs. Viability...
Source: Journal of Biological Chemistry - December 18, 2020 Category: Chemistry Authors: Stephanie J. Guiney, Paul A. Adlard, Peng Lei, Celeste H. Mawal, Ashley I. Bush, David I. Finkelstein, Scott Ayton Tags: Neurobiology Source Type: research
Novel fluorescent GPCR biosensor detects retinal equilibrium binding to opsin and active G protein and arrestin signaling conformations [Molecular Biophysics]
Rhodopsin is a canonical class A photosensitive G protein–coupled receptor (GPCR), yet relatively few pharmaceutical agents targeting this visual receptor have been identified, in part due to the unique characteristics of its light-sensitive, covalently bound retinal ligands. Rhodopsin becomes activated when light isomerizes 11-cis-retinal into an agonist, all-trans-retinal (ATR), which enables the receptor to activate its G protein. We have previously demonstrated that, despite being covalently bound, ATR can display properties of equilibrium binding, yet how this is accomplished is unknown. Here, we describe a new appr...
Source: Journal of Biological Chemistry - December 18, 2020 Category: Chemistry Authors: Christopher T. Schafer, Anthony Shumate, David L. Farrens Tags: Signal Transduction Source Type: research
Shared requirements for key residues in the antibiotic resistance enzymes ErmC and ErmE suggest a common mode of RNA recognition [Enzymology]
Erythromycin-resistance methyltransferases are SAM dependent Rossmann fold methyltransferases that convert A2058 of 23S rRNA to m6 2A2058. This modification sterically blocks binding of several classes of antibiotics to 23S rRNA, resulting in a multidrug-resistant phenotype in bacteria expressing the enzyme. ErmC is an erythromycin resistance methyltransferase found in many Gram-positive pathogens, whereas ErmE is found in the soil bacterium that biosynthesizes erythromycin. Whether ErmC and ErmE, which possess only 24% sequence identity, use similar structural elements for rRNA substrate recognition and positioning is not...
Source: Journal of Biological Chemistry - December 18, 2020 Category: Chemistry Authors: Sebastian J. Rowe, Ryan J. Mecaskey, Mohamed Nasef, Rachel C. Talton, Rory E. Sharkey, Joshua C. Halliday, Jack A. Dunkle Tags: RNA Source Type: research
