To be there when the picture is being painted [Protein Structure and Folding]
This article recalls 1) how I got to be a biochemist; 2) my contributions as an educator and researcher, especially regarding meprin metalloproteases; and 3) my participation in communities of science. Perhaps my reflections will help an aspiring scientist see how fulfilling a career in science can be. (Source: Journal of Biological Chemistry)
Source: Journal of Biological Chemistry - November 20, 2020 Category: Chemistry Authors: Judith S. Bond Tags: Reflections Source Type: research
Structure and kinetics of indole-3-glycerol phosphate synthase from Pseudomonas aeruginosa: Decarboxylation is not essential for indole formation [Protein Structure and Folding]
In tryptophan biosynthesis, the reaction catalyzed by the enzyme indole-3-glycerol phosphate synthase (IGPS) starts with a condensation step in which the substrate's carboxylated phenyl group makes a nucleophilic attack to form the pyrrole ring of the indole, followed by a decarboxylation that restores the aromaticity of the phenyl. IGPS from Pseudomonas aeruginosa has the highest turnover number of all characterized IGPS enzymes, providing an excellent model system to test the necessity of the decarboxylation step. Since the 1960s, this step has been considered to be mechanistically essential based on studies of the IGPSâ...
Source: Journal of Biological Chemistry - November 20, 2020 Category: Chemistry Authors: Annika Soderholm, Matilda S. Newton, Wayne M. Patrick, Maria Selmer Tags: Enzymology Source Type: research
Hoogsteen base pairs increase the susceptibility of double-stranded DNA to cytotoxic damage [Methods and Resources]
As the Watson–Crick faces of nucleobases are protected in dsDNA, it is commonly assumed that deleterious alkylation damage to the Watson–Crick faces of nucleobases predominantly occurs when DNA becomes single-stranded during replication and transcription. However, damage to the Watson–Crick faces of nucleobases has been reported in dsDNA in vitro through mechanisms that are not understood. In addition, the extent of protection from methylation damage conferred by dsDNA relative to ssDNA has not been quantified. Watson–Crick base pairs in dsDNA exist in dynamic equilibrium with Hoogsteen base pairs that expose the W...
Source: Journal of Biological Chemistry - November 20, 2020 Category: Chemistry Authors: Yu Xu, Akanksha Manghrani, Bei Liu, Honglue Shi, Uyen Pham, Amy Liu, Hashim M. Al-Hashimi Tags: DNA and Chromosomes Source Type: research
Visualizing a viral genome with contrast variation small angle X-ray scattering [RNA]
Despite the threat to human health posed by some single-stranded RNA viruses, little is understood about their assembly. The goal of this work is to introduce a new tool for watching an RNA genome direct its own packaging and encapsidation by proteins. Contrast variation small-angle X-ray scattering (CV-SAXS) is a powerful tool with the potential to monitor the changing structure of a viral RNA through this assembly process. The proteins, though present, do not contribute to the measured signal. As a first step in assessing the feasibility of viral genome studies, the structure of encapsidated MS2 RNA was exclusively detec...
Source: Journal of Biological Chemistry - November 20, 2020 Category: Chemistry Authors: Josue San Emeterio, Lois Pollack Tags: RNA Source Type: research
Mechanical contribution to muscle thin filament activation [Bioenergetics]
Vertebrate striated muscle thin filaments are thought to be thermodynamically activated in response to an increase in Ca2+ concentration. We tested this hypothesis by measuring time intervals for gliding runs and pauses of individual skeletal muscle thin filaments in cycling myosin motility assays. A classic thermodynamic mechanism predicts that if chemical potential is constant, transitions between runs and pauses of gliding thin filaments will occur at constant rate as given by a Poisson distribution. In this scenario, rate is given by the odds of a pause, and hence, run times between pauses fit an exponential distributi...
Source: Journal of Biological Chemistry - November 20, 2020 Category: Chemistry Authors: Henry G. Zot, P. Bryant Chase, Javier E. Hasbun, Jose R. Pinto Tags: Molecular Biophysics Source Type: research
Simulations of octapeptin-outer membrane interactions reveal conformational flexibility is linked to antimicrobial potency [Molecular Biophysics]
The octapeptins are lipopeptide antibiotics that are structurally similar to polymyxins yet retain activity against polymyxin-resistant Gram-negative pathogens, suggesting they might be used to treat recalcitrant infections. However, the basis of their unique activity is unclear because of the difficulty in generating high-resolution experimental data of the interaction of antimicrobial peptides with lipid membranes. To elucidate these structure–activity relationships, we employed all-atom molecular dynamics simulations with umbrella sampling to investigate the conformational and energetic landscape of octapeptins intera...
Source: Journal of Biological Chemistry - November 20, 2020 Category: Chemistry Authors: Xukai Jiang, Kai Yang, Bing Yuan, Bin Gong, Lin Wan, Nitin A. Patil, James D. Swarbrick, Kade D. Roberts, Falk Schreiber, Lushan Wang, Tony Velkov, Jian Li Tags: Computational Biology Source Type: research
Mode of targeting to the proteasome determines GFP fate [Enzymology]
The ubiquitin–proteasome system is the canonical pathway for protein degradation in eukaryotic cells. GFP is frequently used as a reporter in proteasomal degradation assays. However, there are multiple variants of GFP in use, and these variants have different intrinsic stabilities. Further, there are multiple means by which substrates are targeted to the proteasome, and these differences could also affect the proteasome's ability to unfold and degrade substrates. Herein we investigate how the fate of GFP variants of differing intrinsic stabilities is determined by the mode of targeting to the proteasome. We compared two ...
Source: Journal of Biological Chemistry - November 20, 2020 Category: Chemistry Authors: Christopher Eric Braganca, Daniel Adam Kraut Tags: Protein Synthesis and Degradation Source Type: research
Pif1, RPA, and FEN1 modulate the ability of DNA polymerase {delta} to overcome protein barriers during DNA synthesis [Enzymology]
Successful DNA replication requires carefully regulated mechanisms to overcome numerous obstacles that naturally occur throughout chromosomal DNA. Scattered across the genome are tightly bound proteins, such as transcription factors and nucleosomes, that are necessary for cell function, but that also have the potential to impede timely DNA replication. Using biochemically reconstituted systems, we show that two transcription factors, yeast Reb1 and Tbf1, and a tightly positioned nucleosome, are strong blocks to the strand displacement DNA synthesis activity of DNA polymerase δ. Although the block imparted by Tbf1 can be o...
Source: Journal of Biological Chemistry - November 20, 2020 Category: Chemistry Authors: Melanie A. Sparks, Peter M. Burgers, Roberto Galletto Tags: DNA and Chromosomes Source Type: research
Ascorbic acid enhances low-density lipoprotein receptor expression by suppressing proprotein convertase subtilisin/kexin 9 expression [Molecular Bases of Disease]
Ascorbic acid, a water-soluble antioxidant, regulates various biological processes and is thought to influence cholesterol. However, little is known about the mechanisms underpinning ascorbic acid-mediated cholesterol metabolism. Here, we determined if ascorbic acid can regulate expression of proprotein convertase subtilisin/kexin 9 (PCSK9), which binds low-density lipoprotein receptor (LDLR) leading to its intracellular degradation, to influence low-density lipoprotein (LDL) metabolism. At cellular levels, ascorbic acid inhibited PCSK9 expression in HepG2 and Huh7 cell lines. Consequently, LDLR expression and cellular LDL...
Source: Journal of Biological Chemistry - November 20, 2020 Category: Chemistry Authors: Dandan Wang, Xiaoxiao Yang, Yuanli Chen, Ke Gong, Maoyun Yu, Yongyao Gao, Ximei Wu, Huaqing Hu, Chenzhong Liao, Jihong Han, Yajun Duan Tags: Lipids Source Type: research
Avian eggshell formation reveals a new paradigm for vertebrate mineralization via vesicular amorphous calcium carbonate [Membrane Biology]
Amorphous calcium carbonate (ACC) is an unstable mineral phase, which is progressively transformed into aragonite or calcite in biomineralization of marine invertebrate shells or avian eggshells, respectively. We have previously proposed a model of vesicular transport to provide stabilized ACC in chicken uterine fluid where eggshell mineralization takes place. Herein, we report further experimental support for this model. We confirmed the presence of extracellular vesicles (EVs) using transmission EM and showed high levels of mRNA of vesicular markers in the oviduct segments where eggshell mineralization occurs. We also de...
Source: Journal of Biological Chemistry - November 20, 2020 Category: Chemistry Authors: Lilian Stapane, Nathalie Le Roy, Jacky Ezagal, Aleȷandro B. Rodriguez–Navarro, Valerie Labas, Lucie Combes–Soia, Maxwell T. Hincke, Joel Gautron Tags: Glycobiology and Extracellular Matrices Source Type: research
Role for carbohydrate response element-binding protein (ChREBP) in high glucose-mediated repression of long noncoding RNA Tug1 [RNA]
Long noncoding RNAs (lncRNAs) have been shown to play key roles in a variety of biological activities of the cell. However, less is known about how lncRNAs respond to environmental cues and what transcriptional mechanisms regulate their expression. Studies from our laboratory have shown that the lncRNA Tug1 (taurine upregulated gene 1) is crucial for the progression of diabetic kidney disease, a major microvascular complication of diabetes. Using a combination of proximity labeling with the engineered soybean ascorbate peroxidase (APEX2), ChIP-qPCR, biotin-labeled oligonucleotide pulldown, and classical promoter luciferase...
Source: Journal of Biological Chemistry - November 20, 2020 Category: Chemistry Authors: Jianyin Long, Daniel L. Galvan, Koki Mise, Yashpal S. Kanwar, Li Li, Naravat Poungavrin, Paul A. Overbeek, Benny H. Chang, Farhad R. Danesh Tags: Gene Regulation Source Type: research
A new approach for quantifying epigenetic landscapes [DNA and Chromosomes]
ChIP-Seq is a widespread experimental method for determining the global enrichment of chromatin modifications and genome-associated factors. Whereas it is straightforward to compare the relative genomic distribution of these epigenetic features, researchers have also made efforts to compare their signal strength using external references for normalization. New work now suggests that these “spike-ins” could lead to inaccurate conclusions due to intrinsic issues of the methodology and instead calls for new criteria of experimental reporting that may permit internal standardization when certain parameters are fulfilled. (...
Source: Journal of Biological Chemistry - November 20, 2020 Category: Chemistry Authors: Wolfgang Fischle Tags: Editors ' Picks Highlights Source Type: research
A physical basis for quantitative ChIP-sequencing [Methods and Resources]
We report a significant increase in immunoprecipitation of presumed off-target histone PTMs after inhibitor treatment, a trend predicted by the model but contrary to spike-in–based indications. Our work also identifies a sensitivity issue in spike-in normalization that has not been considered in the literature, placing limitations on its utility and trustworthiness. We call our new approach the sans-spike-in method for quantitative ChIP-sequencing (siQ-ChIP). A number of changes in community practice of ChIP-Seq, data reporting, and analysis are motivated by this work. (Source: Journal of Biological Chemistry)
Source: Journal of Biological Chemistry - November 20, 2020 Category: Chemistry Authors: Bradley M. Dickson, Rochelle L. Tiedemann, Alison A. Chomiak, Evan M. Cornett, Robert M. Vaughan, Scott B. Rothbart Tags: Editors ' Picks Source Type: research
Direct evidence that Ataxin-2 is a translational activator mediating cytoplasmic polyadenylation [RNA]
The RNA-binding protein Ataxin-2 binds to and stabilizes a number of mRNA sequences, including that of the transactive response DNA-binding protein of 43 kDa (TDP-43). Ataxin-2 is additionally involved in several processes requiring translation, such as germline formation, long-term habituation, and circadian rhythm formation. However, it has yet to be unambiguously demonstrated that Ataxin-2 is actually involved in activating the translation of its target mRNAs. Here we provide direct evidence from a polysome profile analysis showing that Ataxin-2 enhances translation of target mRNAs. Our recently established method for t...
Source: Journal of Biological Chemistry - November 20, 2020 Category: Chemistry Authors: Hiroto Inagaki, Nao Hosoda, Hitomi Tsuiji, Shin-ichi Hoshino Tags: Editors ' Picks Source Type: research
A saturating mutagenesis CRISPR-Cas9-mediated functional genomic screen identifies cis- and trans-regulatory elements of Oct4 in murine ESCs [Gene Regulation]
Regulatory elements (REs) consist of enhancers and promoters that occupy a significant portion of the noncoding genome and control gene expression programs either in cis or in trans. Putative REs have been identified largely based on their regulatory features (co-occupancy of ESC-specific transcription factors, enhancer histone marks, and DNase hypersensitivity) in mouse embryonic stem cells (mESCs). However, less has been established regarding their regulatory functions in their native context. We deployed cis- and trans-regulatory elements scanning through saturating mutagenesis and sequencing (ctSCAN-SMS) to target elem...
Source: Journal of Biological Chemistry - November 20, 2020 Category: Chemistry Authors: Matthew C. Canver, Pratibha Tripathi, Michael J. Bullen, Moshe Olshansky, Yogesh Kumar, Lee H. Wong, Stephen J. Turner, Samuel Lessard, Luca Pinello, Stuart H. Orkin, Partha Pratim Das Tags: Editors ' Picks Source Type: research
