Branched-chain ketoacid overload inhibits insulin action in the muscle [Metabolism]
This study provides evidence for FA-mediated regulation of BCAA-catabolizing enzymes and BCKA content and highlights the biological role of BCKAs in regulating muscle insulin signaling and function. (Source: Journal of Biological Chemistry)
Source: Journal of Biological Chemistry - November 13, 2020 Category: Chemistry Authors: Dipsikha Biswas, Khoi T. Dao, Angella Mercer, Andrew M. Cowie, Luke Duffley, Yassine El Hiani, Petra C. Kienesberger, Thomas Pulinilkunnil Tags: Signal Transduction Source Type: research
Structural basis for the interaction of Shiga toxin 2a with a C-terminal peptide of ribosomal P stalk proteins [Protein Structure and Folding]
The principal virulence factor of human pathogenic enterohemorrhagic Escherichia coli is Shiga toxin (Stx). Shiga toxin 2a (Stx2a) is the subtype most commonly associated with severe disease outcomes such as hemorrhagic colitis and hemolytic uremic syndrome. The catalytic A1 subunit (Stx2A1) binds to the conserved elongation factor binding C-terminal domain (CTD) of ribosomal P stalk proteins to inhibit translation. Stx2a holotoxin also binds to the CTD of P stalk proteins because the ribosome-binding site is exposed. We show here that Stx2a binds to an 11-mer peptide (P11) mimicking the CTD of P stalk proteins with low mi...
Source: Journal of Biological Chemistry - November 13, 2020 Category: Chemistry Authors: Michael J. Rudolph, Simon A. Davis, Nilgun E. Tumer, Xiao-Ping Li Tags: Microbiology Source Type: research
A ribonucleotide reductase from Clostridium botulinum reveals distinct evolutionary pathways to regulation via the overall activity site [Protein Structure and Folding]
Ribonucleotide reductase (RNR) is a central enzyme for the synthesis of DNA building blocks. Most aerobic organisms, including nearly all eukaryotes, have class I RNRs consisting of R1 and R2 subunits. The catalytic R1 subunit contains an overall activity site that can allosterically turn the enzyme on or off by the binding of ATP or dATP, respectively. The mechanism behind the ability to turn the enzyme off via the R1 subunit involves the formation of different types of R1 oligomers in most studied species and R1–R2 octamers in Escherichia coli. To better understand the distribution of different oligomerization mechanis...
Source: Journal of Biological Chemistry - November 13, 2020 Category: Chemistry Authors: Markel Martinez–Carranza, Venkateswara Rao Jonna, Daniel Lundin, Margareta Sahlin, Lars–Anders Carlson, Newal Jemal, Martin Hogbom, Britt–Marie Sȷoberg, Pal Stenmark, Anders Hofer Tags: Enzymology Source Type: research
An autoinhibitory role for the GRF zinc finger domain of DNA glycosylase NEIL3 [Protein Structure and Folding]
The NEIL3 DNA glycosylase maintains genome integrity during replication by excising oxidized bases from single-stranded DNA (ssDNA) and unhooking interstrand cross-links (ICLs) at fork structures. In addition to its N-terminal catalytic glycosylase domain, NEIL3 contains two tandem C-terminal GRF-type zinc fingers that are absent in the other NEIL paralogs. ssDNA binding by the GRF–ZF motifs helps recruit NEIL3 to replication forks converged at an ICL, but the nature of DNA binding and the effect of the GRF–ZF domain on catalysis of base excision and ICL unhooking is unknown. Here, we show that the tandem GRF–ZFs of ...
Source: Journal of Biological Chemistry - November 13, 2020 Category: Chemistry Authors: Alyssa A. Rodriguez, Jessica L. Wojtaszek, Briana H. Greer, Tuhin Haldar, Kent S. Gates, R. Scott Williams, Brandt F. Eichman Tags: DNA and Chromosomes Source Type: research
The human mitochondrial enzyme BCO2 exhibits catalytic activity toward carotenoids and apocarotenoids [Metabolism]
The enzyme β-carotene oxygenase 2 (BCO2) converts carotenoids into more polar metabolites. Studies in mammals, fish, and birds revealed that BCO2 controls carotenoid homeostasis and is involved in the pathway for vitamin A production. However, it is controversial whether BCO2 function is conserved in humans, because of a 4-amino acid long insertion caused by a splice acceptor site polymorphism. We here show that human BCO2 splice variants, BCO2a and BCO2b, are expressed as pre-proteins with mitochondrial targeting sequence (MTS). The MTS of BCO2a directed a green fluorescent reporter protein to the mitochondria when expre...
Source: Journal of Biological Chemistry - November 13, 2020 Category: Chemistry Authors: Linda D. Thomas, Sepalika Bandara, Vipulkumar M. Parmar, Ramkumar Srinivasagan, Nimesh Khadka, Marcin Golczak, Philip D. Kiser, Johannes von Lintig Tags: Lipids Source Type: research
Nef homodimers down-regulate SERINC5 by AP-2-mediated endocytosis to promote HIV-1 infectivity [Molecular Bases of Disease]
SERINC5 is a multipass intrinsic membrane protein that suppresses HIV-1 infectivity when incorporated into budding virions. The HIV-1 Nef virulence factor prevents viral incorporation of SERINC5 by triggering its down-regulation from the producer cell membrane through an AP-2–dependent endolysosomal pathway. However, the mechanistic basis for SERINC5 down-regulation by Nef remains elusive. Here we demonstrate that Nef homodimers are important for SERINC5 down-regulation, trafficking to late endosomes, and exclusion from newly synthesized viral particles. Based on previous X-ray crystal structures, we mutated three conser...
Source: Journal of Biological Chemistry - November 13, 2020 Category: Chemistry Authors: Ryan P. Staudt, Thomas E. Smithgall Tags: Microbiology Source Type: research
Lysine acetylation of F-actin decreases tropomyosin-based inhibition of actomyosin activity [Molecular Biophysics]
Recent proteomics studies of vertebrate striated muscle have identified lysine acetylation at several sites on actin. Acetylation is a reversible post-translational modification that neutralizes lysine's positive charge. Positively charged residues on actin, particularly Lys326 and Lys328, are predicted to form critical electrostatic interactions with tropomyosin (Tpm) that promote its binding to filamentous (F)-actin and bias Tpm to an azimuthal location where it impedes myosin attachment. The troponin (Tn) complex also influences Tpm's position along F-actin as a function of Ca2+ to regulate exposure of myosin-binding si...
Source: Journal of Biological Chemistry - November 13, 2020 Category: Chemistry Authors: William Schmidt, Aditi Madan, D. Brian Foster, Anthony Cammarato Tags: Cell Biology Source Type: research
Dissecting the structural and functional roles of a putative metal entry site in encapsulated ferritins [Enzymology]
Encapsulated ferritins belong to the universally distributed ferritin superfamily, whose members function as iron detoxification and storage systems. Encapsulated ferritins have a distinct annular structure and must associate with an encapsulin nanocage to form a competent iron store that is capable of holding significantly more iron than classical ferritins. The catalytic mechanism of iron oxidation in the ferritin family is still an open question because of the differences in organization of the ferroxidase catalytic site and neighboring secondary metal-binding sites. We have previously identified a putative metal-bindin...
Source: Journal of Biological Chemistry - November 13, 2020 Category: Chemistry Authors: Cecilia Piergentili, Jennifer Ross, Didi He, Kelly J. Gallagher, Will A. Stanley, Laurene Adam, C. Logan Mackay, Arnaud Basle, Kevin J. Waldron, David J. Clarke, Jon Marles–Wright Tags: Protein Structure and Folding Source Type: research
C-terminal tail length guides insertion and assembly of membrane proteins [Cell Biology]
A large number of newly synthesized membrane proteins in the endoplasmic reticulum (ER) are assembled into multiprotein complexes, but little is known about the mechanisms required for assembly membrane proteins. It has been suggested that membrane chaperones might exist, akin to the molecular chaperones that stabilize and direct the assembly of soluble protein complexes, but the mechanisms by which these proteins would bring together membrane protein components is unclear. Here, we have identified that the tail length of the C-terminal transmembrane domains (C-TMDs) determines efficient insertion and assembly of membrane ...
Source: Journal of Biological Chemistry - November 13, 2020 Category: Chemistry Authors: Sha Sun, Malaiyalam Mariappan Tags: Protein Synthesis and Degradation Source Type: research
Lysine acetylation regulates the activity of nuclear Pif1 [DNA and Chromosomes]
In Saccharomyces cerevisiae, the Pif1 helicase functions in both nuclear and mitochondrial DNA replication and repair processes, preferentially unwinding RNA:DNA hybrids and resolving G-quadruplex structures. We sought to determine how the various activities of Pif1 are regulated in vivo. Here, we report lysine acetylation of nuclear Pif1 and demonstrate that it influences both Pif1's cellular roles and core biochemical activities. Using Pif1 overexpression toxicity assays, we determined that the acetyltransferase NuA4 and deacetylase Rpd3 are primarily responsible for the dynamic acetylation of nuclear Pif1. MS analysis r...
Source: Journal of Biological Chemistry - November 13, 2020 Category: Chemistry Authors: Onyekachi E. Ononye, Christopher W. Sausen, Lata Balakrishnan, Matthew L. Bochman Tags: DNA and Chromosomes Source Type: research
Acidity and nucleophilic reactivity of glutathione persulfide [Metabolism]
Persulfides (RSSH/RSS−) participate in sulfur trafficking and metabolic processes, and are proposed to mediate the signaling effects of hydrogen sulfide (H2S). Despite their growing relevance, their chemical properties are poorly understood. Herein, we studied experimentally and computationally the formation, acidity, and nucleophilicity of glutathione persulfide (GSSH/GSS−), the derivative of the abundant cellular thiol glutathione (GSH). We characterized the kinetics and equilibrium of GSSH formation from glutathione disulfide and H2S. A pKa of 5.45 for GSSH was determined, which is 3.49 units below that of GSH. The ...
Source: Journal of Biological Chemistry - November 13, 2020 Category: Chemistry Authors: Dayana Benchoam, Jonathan A. Semelak, Ernesto Cuevasanta, Mauricio Mastrogiovanni, Juan S. Grassano, Gerardo Ferrer–Sueta, Ari Zeida, Madia Truȷillo, Matias N. Moller, Dario A. Estrin, Beatriz Alvarez Tags: Enzymology Source Type: research
Do FeS clusters rule bacterial iron regulation? [Enzymology]
For decades, the bacterial ferric uptake regulator (Fur) has been thought to respond to ferrous iron to transcriptionally regulate genes required for balancing iron uptake, storage, and utilization. Because iron binding to Fur has never been confirmed in vivo, the physiological iron-sensing mechanism remains an open question. Fontenot et al. now show that Fur purified from Escherichia coli binds an all-Cys-coordinated [2Fe-2S] cluster. This finding opens the exciting possibility that Fur may join numerous well-studied bacterial, fungal, and mammalian proteins that use FeS clusters for cellular iron regulation. (Source: Jou...
Source: Journal of Biological Chemistry - November 13, 2020 Category: Chemistry Authors: Roland Lill Tags: Editors ' Picks Highlights Source Type: research
Ferric uptake regulator (Fur) reversibly binds a [2Fe-2S] cluster to sense intracellular iron homeostasis in Escherichia coli [Bioenergetics]
The ferric uptake regulator (Fur) is a global transcription factor that regulates intracellular iron homeostasis in bacteria. The current hypothesis states that when the intracellular “free” iron concentration is elevated, Fur binds ferrous iron, and the iron-bound Fur represses the genes encoding for iron uptake systems and stimulates the genes encoding for iron storage proteins. However, the “iron-bound” Fur has never been isolated from any bacteria. Here we report that the Escherichia coli Fur has a bright red color when expressed in E. coli mutant cells containing an elevated intracellular free iron content bec...
Source: Journal of Biological Chemistry - November 13, 2020 Category: Chemistry Authors: Chelsey R. Fontenot, Homyra Tasnim, Kathryn A. Valdes, Codrina V. Popescu, Huangen Ding Tags: Editors ' Picks Source Type: research
A blueprint for academic laboratories to produce SARS-CoV-2 quantitative RT-PCR test kits [RNA]
Widespread testing for the presence of the novel coronavirus severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in individuals remains vital for controlling the COVID-19 pandemic prior to the advent of an effective treatment. Challenges in testing can be traced to an initial shortage of supplies, expertise, and/or instrumentation necessary to detect the virus by quantitative RT-PCR (RT-qPCR), the most robust, sensitive, and specific assay currently available. Here we show that academic biochemistry and molecular biology laboratories equipped with appropriate expertise and infrastructure can replicate commercially...
Source: Journal of Biological Chemistry - November 13, 2020 Category: Chemistry Authors: Samantha J. Mascuch, Sara Fakhretaha–Aval, Jessica C. Bowman, Minh Thu H. Ma, Gwendell Thomas, Bettina Bommarius, Chieri Ito, Liangȷun Zhao, Gary P. Newnam, Kavita R. Matange, Hem R. Thapa, Brett Barlow, Rebecca K. Donegan, Nguyet A. Nguyen, Tags: Editors ' Picks Source Type: research
The palmitoyl acyltransferases ZDHHC5 and ZDHHC8 are uniquely present in DRG axons and control retrograde signaling via the Gp130/JAK/STAT3 pathway [Cell Biology]
Palmitoylation, the modification of proteins with the lipid palmitate, is a key regulator of protein targeting and trafficking. However, knowledge of the roles of specific palmitoyl acyltransferases (PATs), which catalyze palmitoylation, is incomplete. For example, little is known about which PATs are present in neuronal axons, although long-distance trafficking of palmitoyl-proteins is important for axon integrity and for axon-to-soma retrograde signaling, a process critical for axon development and for responses to injury. Identifying axonally targeted PATs might thus provide insights into multiple aspects of axonal biol...
Source: Journal of Biological Chemistry - November 13, 2020 Category: Chemistry Authors: Kaitlin M. Collura, Jingwen Niu, Shaun S. Sanders, Audrey Montersino, Sabrina M. Holland, Gareth M. Thomas Tags: Editors ' Picks Source Type: research
