A chemoprobe tracks its target [Methods and Resources]
Small-molecule inhibitors of histone-modifying enzymes have significant clinical utility for managing diseases such as cancer. These inhibitors are usually identified and monitored through their effects on the gain or loss of specific histone marks. In cells, multiple related enzymes can place or remove a specific mark; therefore, relying on an indirect measure of inhibitor engagement can be misleading. Mascaró et al. describe a luminescence-based ELISA approach that directly monitors binding of inhibitors to the histone lysine demethylase KDM1A. (Source: Journal of Biological Chemistry)
Source: Journal of Biological Chemistry - May 17, 2019 Category: Chemistry Authors: Aseem Z. Ansari Tags: Editors ' Picks Highlights Source Type: research

Chemoprobe-based assays of histone lysine demethylase 1A target occupation enable in vivo pharmacokinetics and pharmacodynamics studies of KDM1A inhibitors [Genomics and Proteomics]
Screening of cellular activity for inhibitors of histone lysine modifiers is most frequently performed indirectly by analyzing changes in the total levels of histone marks targeted by lysine methylases/demethylases. However, inhibition of histone lysine modifiers often leads to local rather than total changes in histone marks. Also, because histone modifications can be modulated by more than one cellular enzyme, it is not always clear whether changes in histone marks are a direct or indirect consequence of the inhibitor treatment applied. Direct assessment of target occupation can provide a useful tool to quantify the frac...
Source: Journal of Biological Chemistry - May 17, 2019 Category: Chemistry Authors: Cristina Mascaro, Alberto Ortega, Elena Carceller, Raquel Ruiz Rodriguez, Filippo Ciceri, Serena Lunardi, Li Yu, Manuel Hilbert, Tamara Maes Tags: Editors ' Picks Source Type: research

Withdrawal: Transformation potential of Ras isoforms correlates with activation of phosphatidylinositol 3-kinase but not ERK. [Withdrawals/Retractions]
This article has been withdrawn by the authors. In Fig. 3A, lanes 4 and 5 were duplicated in the left ERK activity panel, which the authors state was due to an error during figure preparation. In Fig. 3D, lanes 2–5 of the AKT panel were duplicated in lanes 7–10 of the same panel. Because the original data for Fig. 3D could not be found, the authors state that they do not have a definitive means of verifying the data in question in the paper. Although the authors state that they believe the conclusions of the paper were correct, they have decided that the proper action is to withdraw this paper. (Source: Journal...
Source: Journal of Biological Chemistry - May 17, 2019 Category: Chemistry Authors: Weiquan Li, Tianqing Zhu, Kun-Liang Guan Tags: Withdrawals/Retractions Source Type: research

Withdrawal: BRCA1-induced apoptosis involves inactivation of ERK1/2 activities. [Withdrawals/Retractions]
This article has been withdrawn by Ying Yan and Kenneth H. Cowan. John P. Haas, Min Kim, and Magdalene K. Sgagias could not be reached. The first author identified some issues and brought them to the attention of the Journal. Subsequently, some issues were also noted in the article by the Journal. After careful analysis of all of the original data used for preparing the figures in the publication, the following errors were identified: The Actin immunoblot in the original Fig. 1A (identified from MCF-7) was used in the immunoblot for Fig. 6C (ERK1/2, lanes 3–4). In Fig. 2B (left panel, MCF7 cells), the Actin immunoblo...
Source: Journal of Biological Chemistry - May 17, 2019 Category: Chemistry Authors: Ying Yan, John P. Haas, Min Kim, Magdalene K. Sgagias, Kenneth H. Cowan Tags: Withdrawals/Retractions Source Type: research

Regulation of proline-directed kinases and the trans-histone code H3K9me3/H4K20me3 during human myogenesis [Genomics and Proteomics]
We present a system-level analysis of proteome, phosphoproteome, and chromatin state of precursors of muscle cells (myoblasts) differentiating into specialized myotubes. Using stable isotope labeling of amino acids in cell culture and nano-liqud chromatography-mass spectrometry/mass spectrometry, we found that phosphorylation motifs targeted by the kinases protein kinase C, cyclin-dependent kinase, and mitogen-activated protein kinase showed increased phosphorylation during myodifferentiation of LHCN-M2 human skeletal myoblast cell line. Drugs known to inhibit these kinases either promoted (PD0325901 and GW8510) or stalled...
Source: Journal of Biological Chemistry - May 17, 2019 Category: Chemistry Authors: Natarajan V. Bhanu, Simone Sidoli, Zuo-Fei Yuan, Rosalynn C. Molden, Benjamin A. Garcia Tags: Methods and Resources Source Type: research

Two-long terminal repeat (LTR) DNA circles are a substrate for HIV-1 integrase [Enzymology]
In this report, using in vitro experiments with purified proteins and DNAs along with DNA endonuclease and in vivo integration assays, we show that this circularized genome can also be efficiently used as a substrate in HIV-1 integrase-mediated integration both in vitro and in eukaryotic cells. Notably, we demonstrate that the palindrome cleavage occurs via a two-step mechanism leading to a blunt-ended DNA product, followed by a classical 3′-processing reaction; this cleavage leads to integrase-dependent integration, highlighted by a 5-bp duplication of the host genome. Our results suggest that 2-LTRc may constitute ...
Source: Journal of Biological Chemistry - May 17, 2019 Category: Chemistry Authors: Clemence Richetta, Sylvain Thierry, Eloise Thierry, Paul Lesbats, Delphine Lapaillerie, Soundasse Munir, Frederic Subra, Herve Leh, Eric Deprez, Vincent Parissi, Olivier Delelis Tags: Microbiology Source Type: research

Functional complementation reveals that 9 of the 13 human V-ATPase subunits can functionally substitute for their yeast orthologs [Cell Biology]
In this study, to assess the functional relatedness of the yeast and human V-ATPase subunits, we investigated whether human V-ATPase subunits can complement calcium- or pH-sensitive growth, acidification of the vacuolar lumen, assembly of the V-ATPase complex, and protein sorting in yeast mutants lacking the equivalent yeast genes. These assessments revealed that 9 of the 13 human V-ATPase subunits can partially or fully complement the function of the corresponding yeast subunits. Importantly, sequence similarity was not necessarily correlated with functional complementation. We also found that besides all Vo domain subuni...
Source: Journal of Biological Chemistry - May 17, 2019 Category: Chemistry Authors: Michiko Abe, Mayu Saito, Ayana Tsukahara, Shuka Shiokawa, Kazuma Ueno, Hiroki Shimamura, Makoto Nagano, Junko Y. Toshima, Jiro Toshima Tags: Cell Biology Source Type: research

Dynamic and structural differences between heme oxygenase-1 and -2 are due to differences in their C-terminal regions [Enzymology]
Heme oxygenase (HO) catalyzes heme degradation, a process crucial for regulating cellular levels of this vital, but cytotoxic, cofactor. Two HO isoforms, HO1 and HO2, exhibit similar catalytic mechanisms and efficiencies. They also share catalytic core structures, including the heme-binding site. Outside their catalytic cores are two regions unique to HO2: a 20-amino acid–long N-terminal extension and a C-terminal domain containing two heme regulatory motifs (HRMs) that bind heme independently of the core. Both HO isoforms contain a C-terminal hydrophobic membrane anchor; however, their sequences diverge. Here, using...
Source: Journal of Biological Chemistry - May 17, 2019 Category: Chemistry Authors: Brent A. Kochert, Angela S. Fleischhacker, Thomas E. Wales, Donald F. Becker, John R. Engen, Stephen W. Ragsdale Tags: Protein Structure and Folding Source Type: research

Indomethacin impairs mitochondrial dynamics by activating the PKC{zeta}-p38-DRP1 pathway and inducing apoptosis in gastric cancer and normal mucosal cells [Cell Biology]
The subcellular mechanism by which nonsteroidal anti-inflammatory drugs (NSAIDs) induce apoptosis in gastric cancer and normal mucosal cells is elusive because of the diverse cyclooxygenase-independent effects of these drugs. Using human gastric carcinoma cells (AGSs) and a rat gastric injury model, here we report that the NSAID indomethacin activates the protein kinase Cζ (PKCζ)–p38 MAPK (p38)–dynamin-related protein 1 (DRP1) pathway and thereby disrupts the physiological balance of mitochondrial dynamics by promoting mitochondrial hyper-fission and dysfunction leading to apoptosis. Notably, DRP1 kno...
Source: Journal of Biological Chemistry - May 17, 2019 Category: Chemistry Authors: Somnath Mazumder, Rudranil De, Subhashis Debsharma, Samik Bindu, Pallab Maity, Souvik Sarkar, Shubhra Jyoti Saha, Asim Azhar Siddiqui, Chinmoy Banerjee, Shiladitya Nag, Debanjan Saha, Saikat Pramanik, Kalyan Mitra, Uday Bandyopadhyay Tags: Signal Transduction Source Type: research

Novel MASP-2 inhibitors developed via directed evolution of human TFPI1 are potent lectin pathway inhibitors [Molecular Bases of Disease]
The lectin pathway (LP) of the complement system is an important antimicrobial defense mechanism, but it also contributes significantly to ischemia reperfusion injury (IRI) associated with myocardial infarct, stroke, and several other clinical conditions. Mannan-binding lectin–associated serine proteinase 2 (MASP-2) is essential for LP activation, and therefore, it is a potential drug target. We have previously developed the first two generations of MASP-2 inhibitors by in vitro evolution of two unrelated canonical serine proteinase inhibitors. These inhibitors were selective LP inhibitors, but their nonhuman origin ...
Source: Journal of Biological Chemistry - May 17, 2019 Category: Chemistry Authors: David Szakacs, Andrea Kocsis, Robert Szasz, Peter Gal, Gabor Pal Tags: Immunology Source Type: research

(Pro)renin receptor-mediated myocardial injury, apoptosis, and inflammatory response in rats with diabetic cardiomyopathy [Metabolism]
In this study, we hypothesized that PRR is involved in the pathogenesis of DCM and mediates myocardial injury in DCM. To explore the role of PRR in DCM, we evaluated the effects of PRR overexpression and knockdown on the DCM phenotype in vivo and in vitro. The results show that PRR overexpression exacerbates myocardial injury and the inflammatory response in rats with DCM. Conversely, PRR knockdown alleviates myocardial fibrosis, apoptosis, and the inflammatory response, reversing the cardiac dysfunction in rats with DCM. In cell experiments, PRR overexpression also up-regulated the protein expression of collagen I and fib...
Source: Journal of Biological Chemistry - May 17, 2019 Category: Chemistry Authors: Xuefei Dong, Shiran Yu, Ying Wang, Min Yang, Jie Xiong, Naier Hei, Bo Dong, Qing Su, Jing Chen Tags: Molecular Bases of Disease Source Type: research

The kinase PERK and the transcription factor ATF4 play distinct and essential roles in autophagy resulting from tunicamycin-induced ER stress [Signal Transduction]
In conclusion, our results indicate that TM-induced UPR activates functional autophagy, and whereas IRE1 is a negative regulator, PERK and ATF4 are required at distinct steps in the autophagic pathway. (Source: Journal of Biological Chemistry)
Source: Journal of Biological Chemistry - May 17, 2019 Category: Chemistry Authors: Morten Luhr, Maria Lyngaas Torgersen, Paula Szalai, Adnan Hashim, Andreas Brech, Judith Staerk, Nikolai Engedal Tags: Cell Biology Source Type: research

Dephosphorylation of the transcriptional cofactor NACA by the PP1A phosphatase enhances cJUN transcriptional activity and osteoblast differentiation [Signal Transduction]
The transcriptional cofactor nascent polypeptide-associated complex and co-regulator α (NACA) regulates osteoblast maturation and activity. NACA functions, at least in part, by binding to Jun proto-oncogene, AP-1 transcription factor subunit (cJUN) and potentiating the transactivation of AP-1 targets such as osteocalcin (Bglap) and matrix metallopeptidase 9 (Mmp9). NACA activity is modulated by phosphorylation carried out by several kinases, but a phosphatase regulating NACA's activity remains to be identified. Here, we used affinity purification with MS in HEK293T cells to isolate NACA complexes and identified prote...
Source: Journal of Biological Chemistry - May 17, 2019 Category: Chemistry Authors: William N. Addison, Martin Pellicelli, Rene St–Arnaud Tags: Gene Regulation Source Type: research

A small-molecule inhibitor of the DNA recombinase Rad51 from Plasmodium falciparum synergizes with the antimalarial drugs artemisinin and chloroquine [Molecular Bases of Disease]
Malaria parasites repair DNA double-strand breaks (DSBs) primarily through homologous recombination (HR). Here, because the unrepaired DSBs lead to the death of the unicellular parasite Plasmodium falciparum, we investigated its recombinase, PfRad51, as a potential drug target. Undertaking an in silico screening approach, we identified a compound, B02, that docks to the predicted tertiary structure of PfRad51 with high affinity. B02 inhibited a drug-sensitive P. falciparum strain (3D7) and multidrug-resistant parasite (Dd2) in culture, with IC50 values of 8 and 3 μm, respectively. We found that B02 is more potent agains...
Source: Journal of Biological Chemistry - May 17, 2019 Category: Chemistry Authors: Pratap Vydyam, Dibyendu Dutta, Niranjan Sutram, Sunanda Bhattacharyya, Mrinal Kanti Bhattacharyya Tags: DNA and Chromosomes Source Type: research

Covalent modification of Cys-239 in {beta}-tubulin by small molecules as a strategy to promote tubulin heterodimer degradation [Signal Transduction]
Clinical microtubule-targeting drugs are functionally divided into microtubule-destabilizing and microtubule-stabilizing agents. Drugs from both classes achieve microtubule inhibition by binding different sites on tubulin and inhibiting or promoting polymerization with no concomitant effects on the protein levels of tubulin heterodimers. Here, we have identified a series of small molecules with diverse structures potentially representing a third class of novel tubulin inhibitors that promote degradation by covalent binding to Cys-239 of β-tubulin. The small molecules highlighted in this study include T0070907 (a perox...
Source: Journal of Biological Chemistry - May 17, 2019 Category: Chemistry Authors: Jianhong Yang, Yong Li, Wei Yan, Weimin Li, Qiang Qiu, Haoyu Ye, Lijuan Chen Tags: Cell Biology Source Type: research

High-resolution structure of RGS17 suggests a role for Ca2+ in promoting the GTPase-activating protein activity by RZ subfamily members [Protein Structure and Folding]
Regulator of G protein signaling (RGS) proteins are negative regulators of G protein–coupled receptor (GPCR) signaling through their ability to act as GTPase-activating proteins (GAPs) for activated Gα subunits. Members of the RZ subfamily of RGS proteins bind to activated Gαo, Gαz, and Gαi1–3 proteins in the nervous system and thereby inhibit downstream pathways, including those involved in Ca2+-dependent signaling. In contrast to other RGS proteins, little is known about RZ subfamily structure and regulation. Herein, we present the 1.5-Å crystal structure of RGS17, the most compl...
Source: Journal of Biological Chemistry - May 17, 2019 Category: Chemistry Authors: Monita Sieng, Michael P. Hayes, Joseph B. O'Brien, C. Andrew Fowler, Jon C. Houtman, David L. Roman, Angeline M. Lyon Tags: Signal Transduction Source Type: research

Non-canonical ubiquitination of the cholesterol-regulated degron of squalene monooxygenase [Lipids]
Squalene monooxygenase (SM) is a rate-limiting enzyme in cholesterol synthesis. The region comprising the first 100 amino acids, termed SM N100, represents the shortest cholesterol-responsive degron and enables SM to sense excess cholesterol in the endoplasmic reticulum (ER) membrane. Cholesterol accelerates the ubiquitination of SM by membrane-associated ring-CH type finger 6 (MARCH6), a key E3 ubiquitin ligase involved in ER-associated degradation. However, the ubiquitination site required for cholesterol regulation of SM N100 is unknown. Here, we used SM N100 fused to GFP as a model degron to recapitulate cholesterol-me...
Source: Journal of Biological Chemistry - May 17, 2019 Category: Chemistry Authors: Ngee Kiat Chua, Gene Hart-Smith, Andrew J. Brown Tags: Lipids Source Type: research

Human red and green cone opsins are O-glycosylated at an N-terminal Ser/Thr-rich domain conserved in vertebrates [Molecular Bases of Disease]
There are fundamental differences in the structures of outer segments between rod and cone photoreceptor cells in the vertebrate retina. Visual pigments are the only essential membrane proteins that differ between rod and cone outer segments, making it likely that they contribute to these structural differences. Human rhodopsin is N-glycosylated on Asn2 and Asn15, whereas human (h) red and green cone opsins (hOPSR and hOPSG, respectively) are N-glycosylated at Asn34. Here, utilizing a monoclonal antibody (7G8 mAB), we demonstrate that hOPSR and hOPSG from human retina also are O-glycosylated with full occupancy. We determi...
Source: Journal of Biological Chemistry - May 17, 2019 Category: Chemistry Authors: David Salom, Hui Jin, Thomas A. Gerken, Clinton Yu, Lan Huang, Krzysztof Palczewski Tags: Membrane Biology Source Type: research

Flavonoids enhance rod opsin stability, folding, and self-association by directly binding to ligand-free opsin and modulating its conformation [Signal Transduction]
In this study, we used a combination of computational methods, biochemical and biophysical approaches, including bioluminescence resonance energy transfer, and mammalian cell expression systems to clarify the effects of four common bioactive flavonoids (quercetin, myricetin, and their mono-glycosylated forms quercetin-3-rhamnoside and myricetrin) on rod opsin stability, function, and membrane organization. We observed that by directly interacting with ligand-free opsin, flavonoids modulate its conformation, thereby causing faster entry of the retinal chromophore into its binding pocket. Moreover, flavonoids significantly i...
Source: Journal of Biological Chemistry - May 17, 2019 Category: Chemistry Authors: Joseph T. Ortega, Tanu Parmar, Beata Jastrzebska Tags: Protein Structure and Folding Source Type: research

Biochemical characterization of the Lassa virus L protein [Enzymology]
The L protein of arena- and bunyaviruses is structurally and functionally related to the orthomyxovirus polymerase complex. It plays a central role in the viral life cycle, as it replicates the virus genome and generates viral mRNA via a cap-snatching mechanism. Here, we aimed to biochemically characterize the L protein of Lassa virus, a human-pathogenic arenavirus endemic in West Africa. Full-length 250-kDa L protein was expressed using a baculovirus expression system. A low-resolution structure calculated from small-angle X-ray scattering data revealed a conformation similar to that in the crystal structure of the orthom...
Source: Journal of Biological Chemistry - May 17, 2019 Category: Chemistry Authors: Dominik Vogel, Maria Rosenthal, Nadȷa Gogrefe, Sophia Reindl, Stephan Gunther Tags: RNA Source Type: research

A proline insertion-deletion in the spike glycoprotein fusion peptide of mouse hepatitis virus strongly alters neuropathology [Neurobiology]
Fusion peptides (FPs) in spike proteins are key players mediating early events in cell-to-cell fusion, vital for intercellular viral spread. A proline residue located at the central FP region has often been suggested to have a distinctive role in this fusion event. The spike glycoprotein from strain RSA59 (PP) of mouse hepatitis virus (MHV) contains two central, consecutive prolines in the FP. Here, we report that deletion of one of these proline residues, resulting in RSA59 (P), significantly affected neural cell syncytia formation and viral titers postinfection in vitro. Transcranial inoculation of C57Bl/6 mice with RSA5...
Source: Journal of Biological Chemistry - May 17, 2019 Category: Chemistry Authors: Manmeet Singh, Abhinoy Kishore, Dibyajyoti Maity, Punnepalli Sunanda, Bankala Krishnarjuna, Sreeparna Vappala, Srinivasarao Raghothama, Lawrence C. Kenyon, Debnath Pal, Jayasri Das Sarma Tags: Microbiology Source Type: research

Unraveling the structural elements of pH sensitivity and substrate binding in the human zinc transporter SLC39A2 (ZIP2) [Protein Structure and Folding]
The transport and ion-coupling mechanisms of ZIP transporters remain largely uncharacterized. Previous work in our laboratory has revealed that the solute carrier family 39 member A2 (SLC39A2/ZIP2) increases its substrate transport rate in the presence of extracellular H+. Here, we used a combination of in silico and in vitro techniques involving structural modeling, mutagenesis, and functional characterization in HEK293 cells to identify amino acid residues potentially relevant for both the ZIP2–H+ interaction and substrate binding. Our ZIP2 models revealed a cluster of charged residues close to the substrate–...
Source: Journal of Biological Chemistry - May 17, 2019 Category: Chemistry Authors: Gergely Gyimesi, Giuseppe Albano, Daniel G. Fuster, Matthias A. Hediger, Jonai Puȷol–Gimenez Tags: Membrane Biology Source Type: research

Proteomic discovery of substrates of the cardiovascular protease ADAMTS7 [Molecular Bases of Disease]
The protease ADAMTS7 functions in the extracellular matrix (ECM) of the cardiovascular system. However, its physiological substrate specificity and mechanism of regulation remain to be explored. To address this, we conducted an unbiased substrate analysis using terminal amine isotopic labeling of substrates (TAILS). The analysis identified candidate substrates of ADAMTS7 in the human fibroblast secretome, including proteins with a wide range of functions, such as collagenous and noncollagenous extracellular matrix proteins, growth factors, proteases, and cell-surface receptors. It also suggested that autolysis occurs at Gl...
Source: Journal of Biological Chemistry - May 17, 2019 Category: Chemistry Authors: Alain Colige, Christine Monseur, James T. B. Crawley, Salvatore Santamaria, Rens de Groot Tags: Enzymology Source Type: research

Heterologous regulation of CXCR4 lysosomal trafficking [Cell Biology]
G protein–coupled receptor (GPCR) signaling is regulated by members of the protein kinase C (PKC) and GPCR kinase (GRK) families, although the relative contribution of each to GPCR function varies among specific GPCRs. The CXC motif receptor 4 (CXCR4) is a member of the GPCR superfamily that binds the CXC motif chemokine ligand 12 (CXCL12), initiating signaling that is subsequently terminated in part by internalization and lysosomal degradation of CXCR4. The purpose of this study is to define the relative contribution of PKC and GRK to CXCR4 signaling attenuation by studying their effects on CXCR4 lysosomal trafficki...
Source: Journal of Biological Chemistry - May 17, 2019 Category: Chemistry Authors: Adriana Caballero, Sarah A. Mahn, Mudassir S. Ali, M. Rose Rogers, Adriano Marchese Tags: Cell Biology Source Type: research

Active-site differences between substrate-free and ritonavir-bound cytochrome P450 (CYP) 3A5 reveal plasticity differences between CYP3A5 and CYP3A4 [Enzymology]
Cytochrome P450 (CYP) 3A4 is a major contributor to hepatic drug and xenobiotic metabolism in human adults. The related enzyme CYP3A5 is also expressed in adult liver and has broader age and tissue distributions. However, CYP3A5 expression is low in most Caucasians because of the prevalence of an allele that leads to an incorrectly spliced mRNA and premature termination of translation. When expressed, CYP3A5 expands metabolic capabilities and can augment CYP3A4-mediated drug metabolism, thereby reducing drug efficacy and potentially requiring dose adjustments. The extensive role of CYP3A4 in drug metabolism reflects in par...
Source: Journal of Biological Chemistry - May 17, 2019 Category: Chemistry Authors: Mei-Hui Hsu, Eric F. Johnson Tags: Protein Structure and Folding Source Type: research

A catalytic domain variant of mitofusin requiring a wildtype paralog for function uncouples mitochondrial outer-membrane tethering and fusion [Membrane Biology]
Mitofusins (Mfns) are dynamin-related GTPases that mediate mitochondrial outer-membrane fusion, a process that is required for mitochondrial and cellular health. In Mfn1 and Mfn2 paralogs, a conserved phenylalanine (Phe-202 (Mfn1) and Phe-223 (Mfn2)) located in the GTPase domain on a conserved β strand is part of an aromatic network in the core of this domain. To gain insight into the poorly understood mechanism of Mfn-mediated membrane fusion, here we characterize a Mitofusin mutant variant etiologically linked to Charcot–Marie–Tooth syndrome. From analysis of mitochondrial structure in cells and mitochon...
Source: Journal of Biological Chemistry - May 17, 2019 Category: Chemistry Authors: Emily A. Engelhart, Suzanne Hoppins Tags: Cell Biology Source Type: research

The unique trimeric assembly of the virulence factor HtrA from Helicobacter pylori occurs via N-terminal domain swapping [Microbiology]
Knowledge of the molecular mechanisms of specific bacterial virulence factors can significantly contribute to antibacterial drug discovery. Helicobacter pylori is a Gram-negative microaerophilic bacterium that infects almost half of the world's population, leading to gastric disorders and even gastric cancer. H. pylori expresses a series of virulence factors in the host, among which high-temperature requirement A (HpHtrA) is a newly identified serine protease secreted by H. pylori. HpHtrA cleaves the extracellular domain of the epithelial cell surface adhesion protein E-cadherin and disrupts gastric epithelial cell junctio...
Source: Journal of Biological Chemistry - May 17, 2019 Category: Chemistry Authors: Zhemin Zhang, Qi Huang, Xuan Tao, Guobing Song, Peng Zheng, Hongyan Li, Hongzhe Sun, Wei Xia Tags: Protein Structure and Folding Source Type: research

Substrate specificity of the pyrophosphohydrolase LpxH determines the asymmetry of Bordetella pertussis lipid A [Immunology]
Lipopolysaccharides are anchored to the outer membrane of Gram-negative bacteria by a hydrophobic moiety known as lipid A, which potently activates the host innate immune response. Lipid A of Bordetella pertussis, the causative agent of whooping cough, displays unusual structural asymmetry with respect to the length of the acyl chains at the 3 and 3′ positions, which are 3OH-C10 and 3OH-C14 chains, respectively. Both chains are attached by the acyltransferase LpxA, the first enzyme in the lipid A biosynthesis pathway, which, in B. pertussis, has limited chain length specificity. However, this only partially explains ...
Source: Journal of Biological Chemistry - May 17, 2019 Category: Chemistry Authors: Jesus Arenas, Elder Pupo, Eline de Jonge, Jesus Perez–Ortega, Joerg Schaarschmidt, Peter van der Ley, Jan Tommassen Tags: Cell Biology Source Type: research

A chloride ring is an ancient evolutionary innovation mediating the assembly of the collagen IV scaffold of basement membranes [Cell Biology]
Collagen IV scaffold is a principal component of the basement membrane (BM), a specialized extracellular matrix that is essential for animal multicellularity and tissue evolution. Scaffold assembly begins with the trimerization of α-chains into protomers inside the cell, which then are secreted and undergo oligomerization outside the cell. For the ubiquitous scaffold composed of α1- and α2-chains, both intracellular and extracellular stages are mediated by the noncollagenous domain (NC1). The association of protomers is chloride-dependent, whereby chloride ions induce interactions of the protomers' trimer...
Source: Journal of Biological Chemistry - May 17, 2019 Category: Chemistry Authors: Vadim Pedchenko, Ryan Bauer, Elena N. Pokidysheva, Alaa Al-Shaer, Nancy R. Forde, Aaron L. Fidler, Billy G. Hudson, Sergei P. Boudko Tags: Glycobiology and Extracellular Matrices Source Type: research

Correction: Biosynthesis of O-N-acetylgalactosamine glycans in the human cell nucleus. [Additions and Corrections]
VOLUME 294 (2019) PAGES 2997–3011The mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium via the PRIDE partner repository with the dataset identifier PXD013484 and DOI 10.6019/PXD013484. (Source: Journal of Biological Chemistry)
Source: Journal of Biological Chemistry - May 10, 2019 Category: Chemistry Authors: Romina B. Cejas, Virginia Lorenz, Yohana C. Garay, Fernando J. Irazoqui Tags: Additions and Corrections Source Type: research

Correction: Control of mitochondrial superoxide production by reverse electron transport at complex I. [Additions and Corrections]
VOLUME 293 (2018) PAGES 9869–9879In Figs. 4B, 4C, and 7, the units for the y axes were incorrect. These errors have now been corrected and do not affect the results or conclusions of this work.jbc;294/19/7966/F4F1F4Figure 4, B and C.jbc;294/19/7966/F7F2F7Figure 7. (Source: Journal of Biological Chemistry)
Source: Journal of Biological Chemistry - May 10, 2019 Category: Chemistry Authors: Ellen L. Robb, Andrew R. Hall, Tracy A. Prime, Simon Eaton, Marten Szibor, Carlo Viscomi, Andrew M. James, Michael P. Murphy Tags: Additions and Corrections Source Type: research

Identification, characterization, and structural analyses of a fungal endo-{beta}-1,2-glucanase reveal a new glycoside hydrolase family [Protein Structure and Folding]
endo-β-1,2-Glucanase (SGL) is an enzyme that hydrolyzes β-1,2-glucans, which play important physiological roles in some bacteria as a cyclic form. To date, no eukaryotic SGL has been identified. We purified an SGL from Talaromyces funiculosus (TfSGL), a soil fungus, to homogeneity and then cloned the complementary DNA encoding the enzyme. TfSGL shows no significant sequence similarity to any known glycoside hydrolase (GH) families, but shows significant similarity to certain eukaryotic proteins with unknown functions. The recombinant TfSGL (TfSGLr) specifically hydrolyzed linear and cyclic β-1,2-glucans to s...
Source: Journal of Biological Chemistry - May 10, 2019 Category: Chemistry Authors: Nobukiyo Tanaka, Masahiro Nakajima, Megumi Narukawa-Nara, Hiroki Matsunaga, Shinji Kamisuki, Hiroki Aramasa, Yuta Takahashi, Naohisa Sugimoto, Koichi Abe, Tohru Terada, Akimasa Miyanaga, Tetsuro Yamashita, Fumio Sugawara, Takashi Kamakura, Shiro Komba, Hi Tags: Enzymology Source Type: research

Activity and structure of Pseudomonas putida MPE, a manganese-dependent single-strand DNA endonuclease encoded in a nucleic acid repair gene cluster [Enzymology]
A recently identified and widely prevalent prokaryal gene cluster encodes a suite of enzymes with imputed roles in nucleic acid repair. The enzymes are as follows: MPE, a DNA endonuclease; Lhr-Core, a 3′–5′ DNA helicase; LIG, an ATP-dependent DNA ligase; and Exo, a metallo-β-lactamase-family nuclease. Bacterial and archaeal MPE proteins belong to the binuclear metallophosphoesterase superfamily that includes the well-studied DNA repair nucleases Mre11 and SbcD. Here, we report that the Pseudomonas putida MPE protein is a manganese-dependent DNA endonuclease that incises either linear single strands o...
Source: Journal of Biological Chemistry - May 10, 2019 Category: Chemistry Authors: Anam Ejaz, Yehuda Goldgur, Stewart Shuman Tags: DNA and Chromosomes Source Type: research

Tau protein aggregates inhibit the protein-folding and vesicular trafficking arms of the cellular proteostasis network [Cell Biology]
Tauopathies are a diverse class of neurodegenerative diseases characterized by the formation of insoluble tau aggregates and the loss of cellular function and neuronal death. Tau inclusions have been shown to contain a number of proteins, including molecular chaperones, but the consequences of these entrapments are not well established. Here, using a human cell system for seeding-dependent tau aggregation, we demonstrate that the molecular chaperones heat-shock cognate 71-kDa protein (HSC70)/heat-shock protein 70 (HSP70), HSP90, and J-domain co-chaperones are sequestered by tau aggregates. By employing single-cell analysis...
Source: Journal of Biological Chemistry - May 10, 2019 Category: Chemistry Authors: Anan Yu, Susan G. Fox, Annalisa Cavallini, Caroline Kerridge, Michael J. O'Neill, Joanna Wolak, Suchira Bose, Richard I. Morimoto Tags: Molecular Bases of Disease Source Type: research

Mechanism and regulation of ferrous heme-nitric oxide (NO) oxidation in NO synthases [Computational Biology]
Nitric oxide (NO) synthases (NOSs) catalyze the formation of NO from l-arginine. We have shown previously that the NOS enzyme catalytic cycle involves a large number of reactions but can be characterized by a global model with three main rate-limiting steps. These are the rate of heme reduction by the flavin domain (kr), of dissociation of NO from the ferric heme-NO complex (kd), and of oxidation of the ferrous heme-NO complex (kox). The reaction of oxygen with the ferrous heme-NO species is part of a futile cycle that does not directly contribute to NO synthesis but allows a population of inactive enzyme molecules to retu...
Source: Journal of Biological Chemistry - May 10, 2019 Category: Chemistry Authors: Jesus Teȷero, Andrew P. Hunt, Jerome Santolini, Nicolai Lehnert, Dennis J. Stuehr Tags: Enzymology Source Type: research

A photoreactive analog of allopregnanolone enables identification of steroid-binding sites in a nicotinic acetylcholine receptor [Neurobiology]
In this report, we used 11β-(p-azidotetrafluorobenzoyloxy)allopregnanolone (F4N3Bzoxy-AP), a general anesthetic and photoreactive allopregnanolone analog that is a potent GABAAR PAM, to characterize steroid-binding sites in the Torpedo α2βγδ nAChR in its native membrane environment. We found that F4N3Bzoxy-AP (IC50 = 31 μm) is 7-fold more potent than alphaxalone in inhibiting binding of the channel blocker [3H]tenocyclidine to nAChRs in the desensitized state. At 300 μm, neither steroid inhibited binding of [3H]tetracaine, a closed-state selective channel blocker, or of [3H]acetylcholine. ...
Source: Journal of Biological Chemistry - May 10, 2019 Category: Chemistry Authors: Zhiyi Yu, David. C. Chiara, Pavel Y. Savechenkov, Karol S. Bruzik, Jonathan B. Cohen Tags: Protein Structure and Folding Source Type: research

An N-terminal Ca2+-binding motif regulates the secretory pathway Ca2+/Mn2+-transport ATPase SPCA1 [Enzymology]
The Ca2+/Mn2+ transport ATPases 1a and 2 (SPCA1a/2) are closely related to the sarco(endo)plasmic reticulum Ca2+-ATPase (SERCA) and are implicated in breast cancer and Hailey–Hailey skin disease. Here, we purified the human SPCA1a/2 isoforms from a yeast recombinant expression system and compared their biochemical properties after reconstitution. We observed that the purified SPCA1a displays a lower Ca2+ affinity and slightly lower Mn2+ affinity than SPCA2. Remarkably, the turnover rates of SPCA1a in the presence of Mn2+ and SPCA2 incubated with Ca2+ and Mn2+ were comparable, whereas the turnover rate of SPCA1a in Ca...
Source: Journal of Biological Chemistry - May 10, 2019 Category: Chemistry Authors: Jialin Chen, Susanne Smaardijk, Charles-Alexandre Mattelaer, Filip Pamula, Ilse Vandecaetsbeek, Jo Vanoevelen, Frank Wuytack, Eveline Lescrinier, Jan Eggermont, Peter Vangheluwe Tags: Membrane Biology Source Type: research

4-Methylumbelliferyl glucuronide contributes to hyaluronan synthesis inhibition [Cell Biology]
We report here that 4-MUG contributes to HA synthesis inhibition. We observed that oral administration of 4-MUG to mice inhibits HA synthesis, promotes FoxP3+ regulatory T-cell expansion, and prevents autoimmune diabetes. Mice fed either 4-MUG or 4-MU had equivalent 4-MU:4-MUG ratios in serum, liver, and pancreas, indicating that 4-MU and 4-MUG reach an equilibrium in these tissues. LC–tandem MS experiments revealed that 4-MUG is hydrolyzed to 4-MU in serum, thereby greatly increasing the effective bioavailability of 4-MU. Moreover, using intravital 2-photon microscopy, we found that 4-MUG (a nonfluorescent molecule)...
Source: Journal of Biological Chemistry - May 10, 2019 Category: Chemistry Authors: Nadine Nagy, Irina Gurevich, Hedwich F. Kuipers, Shannon M. Ruppert, Payton L. Marshall, Bryan J. Xie, Wenchao Sun, Andrey V. Malkovskiy, Jayakumar Rajadas, Maria Grandoch, Jens W. Fischer, Adam R. Frymoyer, Gernot Kaber, Paul L. Bollyky Tags: Glycobiology and Extracellular Matrices Source Type: research

IL-6 exhibits both cis- and trans-signaling in osteocytes and osteoblasts, but only trans-signaling promotes bone formation and osteoclastogenesis [Signal Transduction]
Interleukin 6 (IL-6) supports development of bone-resorbing osteoclasts by acting early in the osteoblast lineage via membrane-bound (cis) or soluble (trans) receptors. Here, we investigated how IL-6 signals and modifies gene expression in differentiated osteoblasts and osteocytes and determined whether these activities can promote bone formation or support osteoclastogenesis. Moreover, we used a genetically altered mouse with circulating levels of the pharmacological IL-6 trans-signaling inhibitor sgp130-Fc to determine whether IL-6 trans-signaling is required for normal bone growth and remodeling. We found that IL-6 incr...
Source: Journal of Biological Chemistry - May 10, 2019 Category: Chemistry Authors: Narelle E. McGregor, Melissa Murat, Jeevithan Elango, Ingrid J. Poulton, Emma C. Walker, Blessing Crimeen-Irwin, Patricia W. M. Ho, Jonathan H. Gooi, T. John Martin, Natalie A. Sims Tags: Cell Biology Source Type: research

Interaction between the integrin Mac-1 and signal regulatory protein {alpha} (SIRP{alpha}) mediates fusion in heterologous cells [Cell Biology]
Macrophage fusion leading to the formation of multinucleated giant cells is a hallmark of chronic inflammation. Several membrane proteins have been implicated in mediating cell–cell attachment during fusion, but their binding partners remain unknown. Recently, we demonstrated that interleukin-4 (IL-4)-induced fusion of mouse macrophages depends on the integrin macrophage antigen 1 (Mac-1). Surprisingly, the genetic deficiency of intercellular adhesion molecule 1 (ICAM-1), an established ligand of Mac-1, did not impair macrophage fusion, suggesting the involvement of other counter-receptors. Here, using various approa...
Source: Journal of Biological Chemistry - May 10, 2019 Category: Chemistry Authors: Nataly P. Podolnikova, Marketa Hlavackova, Yifei Wu, Valentin P. Yakubenko, James Faust, Arnat Balabiyev, Xu Wang, Tatiana P. Ugarova Tags: Protein Structure and Folding Source Type: research

Methylation deficiency of chromatin proteins is a non-mutational and epigenetic-like trait in evolved lines of the archaeon Sulfolobus solfataricus [Microbiology]
Archaea are a distinct and deeply rooted lineage that harbor eukaryotic-like mechanisms, including several that manage chromosome function. In previous work, the thermoacidophilic crenarchaeon, Sulfolobus solfataricus, was subjected to adaptive laboratory evolution to produce three strains, called SARC, with a new heritable trait of super acid resistance. These strains acquired heritable conserved transcriptomes, yet one strain contained no mutations. Homologous recombination without allele replacement at SARC acid resistance genes caused changes in both phenotype and expression of the targeted gene. As recombination displ...
Source: Journal of Biological Chemistry - May 10, 2019 Category: Chemistry Authors: Tyler Johnson, Sophie Payne, Ryan Grove, Samuel McCarthy, Erin Oeltjen, Collin Mach, Jiri Adamec, Mark A. Wilson, Kevin Van Cott, Paul Blum Tags: Microbiology Source Type: research

Lactic acidosis caused by repressed lactate dehydrogenase subunit B expression down-regulates mitochondrial oxidative phosphorylation via the pyruvate dehydrogenase (PDH)-PDH kinase axis [Cell Biology]
Aerobic glycolysis and mitochondrial dysfunction are key metabolic features of cancer cells, but their interplay during cancer development remains unclear. We previously reported that human hepatoma cells with mitochondrial defects exhibit down-regulated lactate dehydrogenase subunit B (LDHB) expression. Here, using several molecular and biochemical assays and informatics analyses, we investigated how LDHB suppression regulates mitochondrial respiratory activity and contributes to liver cancer progression. We found that transcriptional LDHB down-regulation is an upstream event during suppressed oxidative phosphorylation. W...
Source: Journal of Biological Chemistry - May 10, 2019 Category: Chemistry Authors: Sun Mi Hong, Young-Kyoung Lee, Imkyong Park, So Mee Kwon, Seongki Min, Gyesoon Yoon Tags: Molecular Bases of Disease Source Type: research

Glycosylation of the viral attachment protein of avian coronavirus is essential for host cell and receptor binding [Molecular Bases of Disease]
Avian coronaviruses, including infectious bronchitis virus (IBV), are important respiratory pathogens of poultry. The heavily glycosylated IBV spike protein is responsible for binding to host tissues. Glycosylation sites in the spike protein are highly conserved across viral genotypes, suggesting an important role for this modification in the virus life cycle. Here, we analyzed the N-glycosylation of the receptor-binding domain (RBD) of IBV strain M41 spike protein and assessed the role of this modification in host receptor binding. Ten single Asn–to–Ala substitutions at the predicted N-glycosylation sites of t...
Source: Journal of Biological Chemistry - May 10, 2019 Category: Chemistry Authors: Lisa M. Parsons, Kim M. Bouwman, Hugo Azurmendi, Robert P. de Vries, John F. Cipollo, Monique H. Verheije Tags: Glycobiology and Extracellular Matrices Source Type: research

ELF4 facilitates innate host defenses against Plasmodium by activating transcription of Pf4 and Ppbp [Microbiology]
In conclusion, our study uncovered a distinct role of ELF4, an innate immune molecule, in host defense against malaria. (Source: Journal of Biological Chemistry)
Source: Journal of Biological Chemistry - May 10, 2019 Category: Chemistry Authors: Dandan Wang, Zeming Zhang, Shuang Cui, Yingchi Zhao, Samuel Craft, Erol Fikrig, Fuping You Tags: Immunology Source Type: research

The extracellular domain of epithelial cell adhesion molecule (EpCAM) enhances multipotency of mesenchymal stem cells through EGFR-LIN28-LET7 signaling [Signal Transduction]
Mesenchymal stem cells (MSCs) are widely considered to be an attractive cell source for regenerative therapies, but maintaining multipotency and self-renewal in cultured MSCs is especially challenging. Hence, the development and mechanistic description of strategies that help promote multipotency in MSCs will be vital to future clinical use. Here, using an array of techniques and approaches, including cell biology, RT-quantitative PCR, immunoblotting, immunofluorescence, flow cytometry, and ChIP assays, we show that the extracellular domain of epithelial cell adhesion molecule (EpCAM) (EpEX) significantly increases the lev...
Source: Journal of Biological Chemistry - May 10, 2019 Category: Chemistry Authors: I.-I. Kuan, Chi-Chiu Lee, Chien-Hsu Chen, Jean Lu, Yuan-Sung Kuo, Han-Chung Wu Tags: Cell Biology Source Type: research

Cell surface expression of 78-kDa glucose-regulated protein (GRP78) mediates diabetic nephropathy [Signal Transduction]
In conclusion, our work reveals a role for csGRP78 in HG-induced profibrotic responses in mesangial cells, informing a potential approach to treating diabetic nephropathy. (Source: Journal of Biological Chemistry)
Source: Journal of Biological Chemistry - May 10, 2019 Category: Chemistry Authors: Richard Van Krieken, Neel Mehta, Tony Wang, Mengyu Zheng, Renzhong Li, Bo Gao, Ehab Ayaub, Kjetil Ask, James C. Paton, Adrienne W. Paton, Richard C. Austin, Joan C. Krepinsky Tags: Molecular Bases of Disease Source Type: research

Proteolytic dynamics of human 20S thymoproteasome [Computational Biology]
An efficient immunosurveillance of CD8+ T cells in the periphery depends on positive/negative selection of thymocytes and thus on the dynamics of antigen degradation and epitope production by thymoproteasome and immunoproteasome in the thymus. Although studies in mouse systems have shown how thymoproteasome activity differs from that of immunoproteasome and strongly impacts the T cell repertoire, the proteolytic dynamics and the regulation of human thymoproteasome are unknown. By combining biochemical and computational modeling approaches, we show here that human 20S thymoproteasome and immunoproteasome differ not only in ...
Source: Journal of Biological Chemistry - May 10, 2019 Category: Chemistry Authors: Ulrike Kuckelkorn, Sabine Stubler, Kathrin Textoris–Taube, Christiane Kilian, Agathe Niewienda, Petra Henklein, Katharina Janek, Michael P. H. Stumpf, Michele Mishto, Juliane Liepe Tags: Protein Synthesis and Degradation Source Type: research

Homotypic and heterotypic trans-assembly of human Rab-family small GTPases in reconstituted membrane tethering [Molecular Biophysics]
In conclusion, our findings establish that, in the physiological context, homotypic and heterotypic trans-assemblies of Rab-family small GTPases can provide the essential molecular machinery necessary to drive membrane tethering in eukaryotic endomembrane systems. (Source: Journal of Biological Chemistry)
Source: Journal of Biological Chemistry - May 10, 2019 Category: Chemistry Authors: Kazuya Segawa, Naoki Tamura, Joji Mima Tags: Membrane Biology Source Type: research

Structural and functional analyses of glycoside hydrolase 138 enzymes targeting chain A galacturonic acid in the complex pectin rhamnogalacturonan II [Metabolism]
The metabolism of carbohydrate polymers drives microbial diversity in the human gut microbiome. The selection pressures in this environment have spurred the evolution of a complex reservoir of microbial genes encoding carbohydrate-active enzymes (CAZymes). Previously, we have shown that the human gut bacterium Bacteroides thetaiotaomicron (Bt) can depolymerize the most structurally complex glycan, the plant pectin rhamnogalacturonan II (RGII), commonly found in the human diet. Previous investigation of the RGII-degrading apparatus in Bt identified BT0997 as a new CAZyme family, classified as glycoside hydrolase 138 (GH138)...
Source: Journal of Biological Chemistry - May 10, 2019 Category: Chemistry Authors: Aurore Labourel, Arnaud Basle, Jose Munoz–Munoz, Didier Ndeh, Simon Booth, Sergey A. Nepogodiev, Robert A. Field, Alan Cartmell Tags: Enzymology Source Type: research

Protein arginine methyltransferase 5 (PRMT5) promotes survival of lymphoma cells via activation of WNT/{beta}-catenin and AKT/GSK3{beta} proliferative signaling [Molecular Bases of Disease]
In this report, we investigate the relationship between PRMT5 and WNT/β-CATENIN as well as AKT/GSK3β proliferative signaling in three different types of non-Hodgkin's lymphoma cell lines, clinical samples, and mouse primary lymphoma cells. We show that PRMT5 stimulates WNT/β-CATENIN signaling through direct epigenetic silencing of pathway antagonists, AXIN2 and WIF1, and indirect activation of AKT/GSK3β signaling. PRMT5 inhibition with either shRNA-mediated knockdown or a specific small molecule PRMT5 inhibitor, CMP-5, not only leads to derepression of WNT antagonists and decreased levels of active phos...
Source: Journal of Biological Chemistry - May 10, 2019 Category: Chemistry Authors: Jihyun Chung, Vraȷesh Karkhanis, Robert A. Baiocchi, Said Sif Tags: Gene Regulation Source Type: research