Nonspecific DNA binding by P1 ParA determines the distribution of plasmid partition and repressor activities [Microbiology]
The faithful segregation, or “partition,” of many low-copy number bacterial plasmids is driven by plasmid-encoded ATPases that are represented by the P1 plasmid ParA protein. ParA binds to the bacterial nucleoid via an ATP-dependent nonspecific DNA (nsDNA)-binding activity, which is essential for partition. ParA also has a site-specific DNA-binding activity to the par operator (parOP), which requires either ATP or ADP, and which is essential for it to act as a transcriptional repressor but is dispensable for partition. Here we examine how DNA binding by ParA contributes to the relative distribution of its plasmid parti...
Source: Journal of Biological Chemistry - December 11, 2020 Category: Chemistry Authors: Jamie C. Baxter, William G. Waples, Barbara E. Funnell Tags: DNA and Chromosomes Source Type: research
Leptin modulates pancreatic {beta}-cell membrane potential through Src kinase-mediated phosphorylation of NMDA receptors [Membrane Biology]
The adipocyte-derived hormone leptin increases trafficking of KATP and Kv2.1 channels to the pancreatic β-cell surface, resulting in membrane hyperpolarization and suppression of insulin secretion. We have previously shown that this effect of leptin is mediated by the NMDA subtype of glutamate receptors (NMDARs). It does so by potentiating NMDAR activity, thus enhancing Ca2+ influx and the ensuing downstream signaling events that drive channel trafficking to the cell surface. However, the molecular mechanism by which leptin potentiates NMDARs in β-cells remains unknown. Here, we report that leptin augments NMDAR function...
Source: Journal of Biological Chemistry - December 11, 2020 Category: Chemistry Authors: Veronica A. Cochrane, Yi Wu, Zhongying Yang, Assmaa ElSheikh, Jeremy Dunford, Paul Kievit, Dale A. Fortin, Show-Ling Shyng Tags: Signal Transduction Source Type: research
Optimized incorporation of an unnatural fluorescent amino acid affords measurement of conformational dynamics governing high-fidelity DNA replication [DNA and Chromosomes]
DNA polymerase from bacteriophage T7 undergoes large, substrate-induced conformational changes that are thought to account for high replication fidelity, but prior studies were adversely affected by mutations required to construct a Cys-lite variant needed for site-specific fluorescence labeling. Here we have optimized the direct incorporation of a fluorescent un-natural amino acid, (7-hydroxy-4-coumarin-yl)-ethylglycine, using orthogonal amber suppression machinery in Escherichia coli. MS methods verify that the unnatural amino acid is only incorporated at one position with minimal background. We show that the single fluo...
Source: Journal of Biological Chemistry - December 11, 2020 Category: Chemistry Authors: Tyler L. Dangerfield, Kenneth A. Johnson Tags: Enzymology Source Type: research
Kinetic investigation of the polymerase and exonuclease activities of human DNA polymerase ϵ holoenzyme [DNA and Chromosomes]
In eukaryotic DNA replication, DNA polymerase ε (Polε) is responsible for leading strand synthesis, whereas DNA polymerases α and δ synthesize the lagging strand. The human Polε (hPolε) holoenzyme is comprised of the catalytic p261 subunit and the noncatalytic p59, p17, and p12 small subunits. So far, the contribution of the noncatalytic subunits to hPolε function is not well understood. Using pre-steady-state kinetic methods, we established a minimal kinetic mechanism for DNA polymerization and editing catalyzed by the hPolε holoenzyme. Compared with the 140-kDa N-terminal catalytic fragment of p261 (p261N), which...
Source: Journal of Biological Chemistry - December 11, 2020 Category: Chemistry Authors: Walter J. Zahurancik, Zucai Suo Tags: Enzymology Source Type: research
Identification of a domain critical for Staphylococcus aureus LukED receptor targeting and lysis of erythrocytes [Molecular Bases of Disease]
Leukocidin ED (LukED) is a pore-forming toxin produced by Staphylococcus aureus, which lyses host cells and promotes virulence of the bacteria. LukED enables S. aureus to acquire iron by lysing erythrocytes, which depends on targeting the host receptor Duffy antigen receptor for chemokines (DARC). The toxin also targets DARC on the endothelium, contributing to the lethality observed during bloodstream infection in mice. LukED is comprised of two monomers: LukE and LukD. LukE binds to DARC and facilitates hemolysis, but the closely related Panton–Valentine leukocidin S (LukS-PV) does not bind to DARC and is not hemolytic....
Source: Journal of Biological Chemistry - December 11, 2020 Category: Chemistry Authors: Marilyn T. Vasquez, Ashira Lubkin, Tamara Reyes-Robles, Christopher J. Day, Keenan A. Lacey, Michael P. Jennings, Victor J. Torres Tags: Microbiology Source Type: research
Heme oxygenase-2 is post-translationally regulated by heme occupancy in the catalytic site [Protein Structure and Folding]
We report that, in contrast to other HRM-containing proteins, the cellular protein level and degradation rate of HO2 are independent of heme binding to the HRMs. Rather, under heme deficiency, loss of heme binding to the catalytic site destabilizes HO2. Consistently, an HO2 catalytic site variant that is unable to bind heme exhibits a constant low protein level and an enhanced protein degradation rate compared with the WT HO2. Finally, HO2 is degraded by the lysosome through chaperone-mediated autophagy, distinct from other HRM-containing proteins and HO1, which are degraded by the proteasome. These results reveal a novel ...
Source: Journal of Biological Chemistry - December 11, 2020 Category: Chemistry Authors: Liu Liu, Arti B. Dumbrepatil, Angela S. Fleischhacker, E. Neil G. Marsh, Stephen W. Ragsdale Tags: Protein Synthesis and Degradation Source Type: research
Functional impact of a congenital stationary night blindness type 2 mutation depends on subunit composition of Cav1.4 Ca2+ channels [Neurobiology]
Voltage-gated Cav1 and Cav2 Ca2+ channels are comprised of a pore-forming α1 subunit (Cav1.1-1.4, Cav2.1-2.3) and auxiliary β (β1-4) and α2δ (α2δ−1−4) subunits. The properties of these channels vary with distinct combinations of Cav subunits and alternative splicing of the encoding transcripts. Therefore, the impact of disease-causing mutations affecting these channels may depend on the identities of Cav subunits and splice variants. Here, we analyzed the effects of a congenital stationary night blindness type 2 (CSNB2)-causing mutation, I745T (IT), in Cav1.4 channels typical of those in human retina: Cav1.4 spl...
Source: Journal of Biological Chemistry - December 11, 2020 Category: Chemistry Authors: Brittany Williams, Josue A. Lopez, J. Wesley Maddox, Amy Lee Tags: Membrane Biology Source Type: research
Iron-mediated degradation of ribosomes under oxidative stress is attenuated by manganese [Cell Biology]
We report that Fe2+ promotes degradation of all rRNA species of the yeast ribosome and that it is bound directly to RNA molecules. Furthermore, we demonstrate that Mn2+ competes with Fe2+ for rRNA-binding sites and that protection of ribosomes from Fe2+-mediated rRNA hydrolysis correlates with the restoration of cell viability. Our data, therefore, suggest a relationship between these two transition metals in controlling ribosome stability under oxidative stress. (Source: Journal of Biological Chemistry)
Source: Journal of Biological Chemistry - December 11, 2020 Category: Chemistry Authors: Daniel G. J. Smethurst, Nikolay Kovalev, Erica R. McKenzie, Dimitri G. Pestov, Natalia Shcherbik Tags: RNA Source Type: research
The Arg-293 of Cryptochrome1 is responsible for the allosteric regulation of CLOCK-CRY1 binding in circadian rhythm [Computational Biology]
Mammalian circadian clocks are driven by transcription/translation feedback loops composed of positive transcriptional activators (BMAL1 and CLOCK) and negative repressors (CRYPTOCHROMEs (CRYs) and PERIODs (PERs)). CRYs, in complex with PERs, bind to the BMAL1/CLOCK complex and repress E-box–driven transcription of clock-associated genes. There are two individual CRYs, with CRY1 exhibiting higher affinity to the BMAL1/CLOCK complex than CRY2. It is known that this differential binding is regulated by a dynamic serine-rich loop adjacent to the secondary pocket of both CRYs, but the underlying features controlling loop dyn...
Source: Journal of Biological Chemistry - December 11, 2020 Category: Chemistry Authors: Seref Gul, Cihan Aydin, Onur Ozcan, Berke Gurkan, Saliha Surme, Ibrahim Baris, Ibrahim Halil Kavakli Tags: Gene Regulation Source Type: research
Dysregulation of hsa-miR-34a and hsa-miR-449a leads to overexpression of PACS-1 and loss of DNA damage response (DDR) in cervical cancer [Cell Biology]
We have observed overexpression of PACS-1, a cytosolic sorting protein in primary cervical tumors. Absence of exonic mutations and overexpression at the RNA level suggested a transcriptional and/or posttranscriptional regulation. University of California Santa Cruz genome browser analysis of PACS-1 micro RNAs (miR), revealed two 8-base target sequences at the 3′ terminus for hsa-miR-34a and hsa-miR-449a. Quantitative RT-PCR and Northern blotting studies showed reduced or loss of expression of the two microRNAs in cervical cancer cell lines and primary tumors, indicating dysregulation of these two microRNAs in cervical ca...
Source: Journal of Biological Chemistry - December 11, 2020 Category: Chemistry Authors: Mysore S. Veena, Santanu Raychaudhuri, Saroj K. Basak, Natarajan Venkatesan, Parameet Kumar, Roopa Biswas, Rita Chakrabarti, Jing Lu, Trent Su, Marcus Gallagher-Jones, Marco Morselli, Haiqing Fu, Matteo Pellegrini, Theodore Goldstein, Mirit I. Aladjem, Ma Tags: Molecular Bases of Disease Source Type: research
The glucose-sensing transcription factor ChREBP is targeted by proline hydroxylation [Metabolism]
Cellular energy demands are met by uptake and metabolism of nutrients like glucose. The principal transcriptional regulator for adapting glycolytic flux and downstream pathways like de novo lipogenesis to glucose availability in many cell types is carbohydrate response element–binding protein (ChREBP). ChREBP is activated by glucose metabolites and post-translational modifications, inducing nuclear accumulation and regulation of target genes. Here we report that ChREBP is modified by proline hydroxylation at several residues. Proline hydroxylation targets both ectopically expressed ChREBP in cells and endogenous ChREBP i...
Source: Journal of Biological Chemistry - December 11, 2020 Category: Chemistry Authors: Steffi Heidenreich, Pamela Weber, Heike Stephanowitz, Konstantin M. Petricek, Till Schutte, Moritz Oster, Antti M. Salo, Miriam Knauer, Isabel Goehring, Na Yang, Nicole Witte, Anne Schumann, Manuela Sommerfeld, Matthias Muenzner, Johanna Myllyharȷu Tags: Gene Regulation Source Type: research
Representative cancer-associated U2AF2 mutations alter RNA interactions and splicing [Molecular Bases of Disease]
High-throughput sequencing of hematologic malignancies and other cancers has revealed recurrent mis-sense mutations of genes encoding pre-mRNA splicing factors. The essential splicing factor U2AF2 recognizes a polypyrimidine-tract splice-site signal and initiates spliceosome assembly. Here, we investigate representative, acquired U2AF2 mutations, namely N196K or G301D amino acid substitutions associated with leukemia or solid tumors, respectively. We determined crystal structures of the wild-type (WT) compared with N196K- or G301D-substituted U2AF2 proteins, each bound to a prototypical AdML polypyrimidine tract, at 1.5, 1...
Source: Journal of Biological Chemistry - December 11, 2020 Category: Chemistry Authors: Debanjana Maji, Eliezra Glasser, Steven Henderson, Justin Galardi, Mary J. Pulvino, Jermaine L. Jenkins, Clara L. Kielkopf Tags: Protein Structure and Folding Source Type: research
Microtubule affinity-regulating kinase 4 with an Alzheimer's disease-related mutation promotes tau accumulation and exacerbates neurodegeneration [Neurobiology]
Accumulation of the microtubule-associated protein tau is associated with Alzheimer's disease (AD). In AD brain, tau is abnormally phosphorylated at many sites, and phosphorylation at Ser-262 and Ser-356 plays critical roles in tau accumulation and toxicity. Microtubule affinity–regulating kinase 4 (MARK4) phosphorylates tau at those sites, and a double de novo mutation in the linker region of MARK4, ΔG316E317D, is associated with an elevated risk of AD. However, it remains unclear how this mutation affects phosphorylation, aggregation, and accumulation of tau and tau-induced neurodegeneration. Here, we report that MARK...
Source: Journal of Biological Chemistry - December 11, 2020 Category: Chemistry Authors: Toshiya Oba, Taro Saito, Akiko Asada, Sawako Shimizu, Koichi M. Iijima, Kanae Ando Tags: Molecular Bases of Disease Source Type: research
M8R tropomyosin mutation disrupts actin binding and filament regulation: The beginning affects the middle and end [Molecular Bases of Disease]
Dilated cardiomyopathy (DCM) is associated with mutations in cardiomyocyte sarcomeric proteins, including α-tropomyosin. In conjunction with troponin, tropomyosin shifts to regulate actomyosin interactions. Tropomyosin molecules overlap via tropomyosin–tropomyosin head-to-tail associations, forming a continuous strand along the thin filament. These associations are critical for propagation of tropomyosin's reconfiguration along the thin filament and key for the cooperative switching between heart muscle contraction and relaxation. Here, we tested perturbations in tropomyosin structure, biochemistry, and function caused ...
Source: Journal of Biological Chemistry - December 11, 2020 Category: Chemistry Authors: Alice Ward Racca, Michael J. Rynkiewicz, Nicholas LaFave, Anita Ghosh, William Lehman, Jeffrey R. Moore Tags: Molecular Bases of Disease Source Type: research
Amyloid precursor protein is a restriction factor that protects against Zika virus infection in mammalian brains [Gene Regulation]
Zika virus (ZIKV) is a neurotropic flavivirus that causes several diseases including birth defects such as microcephaly. Intrinsic immunity is known to be a frontline defense against viruses through host anti-viral restriction factors. Limited knowledge is available on intrinsic immunity against ZIKV in brains. Amyloid precursor protein (APP) is predominantly expressed in brains and implicated in the pathogenesis of Alzheimer's diseases. We have found that ZIKV interacts with APP, and viral infection increases APP expression via enhancing protein stability. Moreover, we identified the viral peptide, HGSQHSGMIVNDTGHETDENRAK...
Source: Journal of Biological Chemistry - December 11, 2020 Category: Chemistry Authors: Amy Lingel, Haishuang Lin, Yuval Gavriel, Eric Weaver, Pascal Polepole, Virginia Lopez, Yuguo Lei, Thomas M. Petro, Beka Solomon, Chi Zhang, Luwen Zhang Tags: Microbiology Source Type: research
