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Journal of Biological Chemistry
Journal Prestige (SJR): 2.672
Citation Impact (citeScore): 4
Number of Followers: 204  
 
  Full-text available via subscription Subscription journal
ISSN (Print) 0021-9258 - ISSN (Online) 1083-351X
Published by ASBMB Homepage  [3 journals]
  • Redistribution of SERCA calcium pump conformers during intracellular
           calcium signaling [Molecular Biophysics]
    • Authors: Olga N. Raguimova; Nikolai Smolin, Elisa Bovo, Siddharth Bhayani, Joseph M. Autry, Aleksey V. Zima, Seth L. Robia
      Pages: 10843 - 10856
      Abstract: The conformational changes of a calcium transport ATPase were investigated with molecular dynamics (MD) simulations as well as fluorescence resonance energy transfer (FRET) measurements to determine the significance of a discrete structural element for regulation of the conformational dynamics of the transport cycle. Previous MD simulations indicated that a loop in the cytosolic domain of the SERCA calcium transporter facilitates an open-to-closed structural transition. To investigate the significance of this structural element, we performed additional MD simulations and new biophysical measurements of SERCA structure and function. Rationally designed in silico mutations of three acidic residues of the loop decreased SERCA domain–domain contacts and increased domain–domain separation distances. Principal component analysis of MD simulations suggested decreased sampling of compact conformations upon N-loop mutagenesis. Deficits in headpiece structural dynamics were also detected by measuring intramolecular FRET of a Cer–YFP–SERCA construct (2-color SERCA). Compared with WT, the mutated 2-color SERCA shows a partial FRET response to calcium, whereas retaining full responsiveness to the inhibitor thapsigargin. Functional measurements showed that the mutated transporter still hydrolyzes ATP and transports calcium, but that maximal enzyme activity is reduced while maintaining similar calcium affinity. In live cells, calcium elevations resulted in concomitant FRET changes as the population of WT 2-color SERCA molecules redistributed among intermediates of the transport cycle. Our results provide novel insights on how the population of SERCA pumps responds to dynamic changes in intracellular calcium.
      PubDate: 2018-07-13T00:10:27-07:00
      DOI: 10.1074/jbc.RA118.002472
      Issue No: Vol. 293, No. 28 (2018)
       
  • Oxidative decarboxylation of pyruvate by 1-deoxy-d-xyulose 5-phosphate
           synthase, a central metabolic enzyme in bacteria [Molecular Biophysics]
    • Authors: Alicia A. DeColli; Natalia S. Nemeria, Ananya Majumdar, Gary J. Gerfen, Frank Jordan, Caren L. Freel Meyers
      Pages: 10857 - 10869
      Abstract: The underexploited antibacterial target 1-deoxy-d-xyluose 5-phosphate (DXP) synthase catalyzes the thiamin diphosphate (ThDP)-dependent formation of DXP from pyruvate and d-glyceraldehyde 3-phosphate (d-GAP). DXP is an essential intermediate in the biosynthesis of ThDP, pyridoxal phosphate, and isoprenoids in many pathogenic bacteria. DXP synthase catalyzes a distinct mechanism in ThDP decarboxylative enzymology in which the first enzyme-bound pre-decarboxylation intermediate, C2α-lactyl-ThDP (LThDP), is stabilized by DXP synthase in the absence of d-GAP, and d-GAP then induces efficient LThDP decarboxylation. Despite the observed LThDP accumulation and lack of evidence for C2α-carbanion formation in the absence of d-GAP, CO2 is released at appreciable levels under these conditions. Here, seeking to resolve these conflicting observations, we show that DXP synthase catalyzes the oxidative decarboxylation of pyruvate under conditions in which LThDP accumulates. O2-dependent LThDP decarboxylation led to one-electron transfer from the C2α-carbanion/enamine to O2, with intermediate ThDP-enamine radical formation, followed by peracetic acid formation en route to acetate. Thus, LThDP formation and decarboxylation and DXP formation were studied under anaerobic conditions. Our results support a model in which O2-dependent LThDP decarboxylation and peracetic acid formation occur in the absence of d-GAP, decreasing the levels of pyruvate and O2 in solution. The relative pyruvate and O2 concentrations then dictate the extent of LThDP accumulation, and its buildup can be observed when [pyruvate]> [O2]. The finding that O2 acts as a structurally distinct trigger of LThDP decarboxylation supports the hypothesis that a mechanism involving small molecule–dependent LThDP decarboxylation equips DXP synthase for diverse, yet uncharacterized cellular functions.
      PubDate: 2018-07-13T00:10:27-07:00
      DOI: 10.1074/jbc.RA118.001980
      Issue No: Vol. 293, No. 28 (2018)
       
  • Ethanolamine ameliorates mitochondrial dysfunction in
           cardiolipin-deficient yeast cells [Bioenergetics]
    • Authors: Writoban Basu Ball; Charli D. Baker, John K. Neff, Gabriel L. Apfel, Kim A. Lagerborg, Gašper Žun, Uroš Petrovič, Mohit Jain, Vishal M. Gohil
      Pages: 10870 - 10883
      Abstract: Cardiolipin (CL) is a signature phospholipid of the mitochondria required for the formation of mitochondrial respiratory chain (MRC) supercomplexes. The destabilization of MRC supercomplexes is the proximal cause of the pathology associated with the depletion of CL in patients with Barth syndrome. Thus, promoting supercomplex formation could ameliorate mitochondrial dysfunction associated with CL depletion. However, to date, physiologically relevant small-molecule regulators of supercomplex formation have not been identified. Here, we report that ethanolamine (Etn) supplementation rescues the MRC defects by promoting supercomplex assembly in a yeast model of Barth syndrome. We discovered this novel role of Etn while testing the hypothesis that elevating mitochondrial phosphatidylethanolamine (PE), a phospholipid suggested to overlap in function with CL, could compensate for CL deficiency. We found that the Etn supplementation rescues the respiratory growth of CL-deficient Saccharomyces cerevisiae cells in a dose-dependent manner but independently of its incorporation into PE. The rescue was specifically dependent on Etn but not choline or serine, the other phospholipid precursors. Etn improved mitochondrial function by restoring the expression of MRC proteins and promoting supercomplex assembly in CL-deficient cells. Consistent with this mechanism, overexpression of Cox4, the MRC complex IV subunit, was sufficient to promote supercomplex formation in CL-deficient cells. Taken together, our work identifies a novel role of a ubiquitous metabolite, Etn, in attenuating mitochondrial dysfunction caused by CL deficiency.
      PubDate: 2018-07-13T00:10:27-07:00
      DOI: 10.1074/jbc.RA118.004014
      Issue No: Vol. 293, No. 28 (2018)
       
  • Inhibition of protein arginine methyltransferase 5 enhances hepatic
           mitochondrial biogenesis [Metabolism]
    • Authors: Lei Huang; Jehnan Liu, Xiao-Ou Zhang, Katelyn Sibley, Sonia M. Najjar, Mary M. Lee, Qiong Wu
      Pages: 10884 - 10894
      Abstract: Protein arginine methyltransferase 5 (PRMT5) regulates gene expression either transcriptionally by symmetric dimethylation of arginine residues on histones H4R3, H3R8, and H2AR3 or at the posttranslational level by methylation of nonhistone target proteins. Although emerging evidence suggests that PRMT5 functions as an oncogene, its role in metabolic diseases is not well-defined. We investigated the role of PRMT5 in promoting high-fat–induced hepatic steatosis. A high-fat diet up-regulated PRMT5 levels in the liver but not in other metabolically relevant tissues such as skeletal muscle or white and brown adipose tissue. This was associated with repression of master transcription regulators involved in mitochondrial biogenesis. In contrast, lentiviral short hairpin RNA–mediated reduction of PRMT5 significantly decreased phosphatidylinositol 3-kinase/AKT signaling in mouse AML12 liver cells. PRMT5 knockdown or knockout decreased basal AKT phosphorylation but boosted the expression of peroxisome proliferator–activated receptor α (PPARα) and PGC-1α with a concomitant increase in mitochondrial biogenesis. Moreover, by overexpressing an exogenous WT or enzyme-dead mutant PRMT5 or by inhibiting PRMT5 enzymatic activity with a small-molecule inhibitor, we demonstrated that the enzymatic activity of PRMT5 is required for regulation of PPARα and PGC-1α expression and mitochondrial biogenesis. Our results suggest that targeting PRMT5 may have therapeutic potential for the treatment of fatty liver.
      PubDate: 2018-07-13T00:10:27-07:00
      DOI: 10.1074/jbc.RA118.002377
      Issue No: Vol. 293, No. 28 (2018)
       
  • Structure-based stabilization of insulin as a therapeutic protein assembly
           via enhanced aromatic-aromatic interactions [Molecular Biophysics]
    • Authors: Nischay K. Rege; Nalinda P. Wickramasinghe, Alisar N. Tustan, Nelson F. B. Phillips, Vivien C. Yee, Faramarz Ismail-Beigi, Michael A. Weiss
      Pages: 10895 - 10910
      Abstract: Key contributions to protein structure and stability are provided by weakly polar interactions, which arise from asymmetric electronic distributions within amino acids and peptide bonds. Of particular interest are aromatic side chains whose directional π-systems commonly stabilize protein interiors and interfaces. Here, we consider aromatic–aromatic interactions within a model protein assembly: the dimer interface of insulin. Semi-classical simulations of aromatic–aromatic interactions at this interface suggested that substitution of residue TyrB26 by Trp would preserve native structure while enhancing dimerization (and hence hexamer stability). The crystal structure of a [TrpB26]insulin analog (determined as a T3Rf3 zinc hexamer at a resolution of 2.25 Å) was observed to be essentially identical to that of WT insulin. Remarkably and yet in general accordance with theoretical expectations, spectroscopic studies demonstrated a 150-fold increase in the in vitro lifetime of the variant hexamer, a critical pharmacokinetic parameter influencing design of long-acting formulations. Functional studies in diabetic rats indeed revealed prolonged action following subcutaneous injection. The potency of the TrpB26-modified analog was equal to or greater than an unmodified control. Thus, exploiting a general quantum-chemical feature of protein structure and stability, our results exemplify a mechanism-based approach to the optimization of a therapeutic protein assembly.
      PubDate: 2018-07-13T00:10:27-07:00
      DOI: 10.1074/jbc.RA118.003650
      Issue No: Vol. 293, No. 28 (2018)
       
  • CHCA-1 is a copper-regulated CTR1 homolog required for normal development,
           copper accumulation, and copper-sensing behavior in Caenorhabditis elegans
           [Cell Biology]
    • Authors: Sai Yuan; Anuj Kumar Sharma, Alexandria Richart, Jaekwon Lee, Byung-Eun Kim
      Pages: 10911 - 10925
      Abstract: Copper plays key roles in catalytic and regulatory biochemical reactions essential for normal growth, development, and health. Dietary copper deficiencies or mutations in copper homeostasis genes can lead to abnormal musculoskeletal development, cognitive disorders, and poor growth. In yeast and mammals, copper is acquired through the activities of the CTR1 family of high-affinity copper transporters. However, the mechanisms of systemic responses to dietary or tissue-specific copper deficiency remain unclear. Here, taking advantage of the animal model Caenorhabditis elegans for studying whole-body copper homeostasis, we investigated the role of a C. elegans CTR1 homolog, CHCA-1, in copper acquisition and in worm growth, development, and behavior. Using sequence homology searches, we identified 10 potential orthologs to mammalian CTR1. Among these genes, we found that chca-1, which is transcriptionally up-regulated in the intestine and hypodermis of C. elegans during copper deficiency, is required for normal growth, reproduction, and maintenance of systemic copper balance under copper deprivation. The intestinal copper transporter CUA-1 normally traffics to endosomes to sequester excess copper, and we found here that loss of chca-1 caused CUA-1 to mislocalize to the basolateral membrane under copper overload conditions. Moreover, animals lacking chca-1 exhibited significantly reduced copper avoidance behavior in response to toxic copper conditions compared with WT worms. These results establish that CHCA-1–mediated copper acquisition in C. elegans is crucial for normal growth, development, and copper-sensing behavior.
      PubDate: 2018-07-13T00:10:27-07:00
      DOI: 10.1074/jbc.RA118.003503
      Issue No: Vol. 293, No. 28 (2018)
       
  • The C-terminal region of the yeast monocarboxylate transporter Jen1 acts
           as a glucose signal-responding degron recognized by the {alpha}-arrestin
           Rod1 [Cell Biology]
    • Authors: Shoki Fujita; Daichi Sato, Hirokazu Kasai, Masataka Ohashi, Shintaro Tsukue, Yutaro Takekoshi, Katsuya Gomi, Takahiro Shintani
      Pages: 10926 - 10936
      Abstract: In response to changes in nutrient conditions, cells rearrange the composition of plasma membrane (PM) transporters to optimize their metabolic flux. Not only transcriptional gene regulation, but also inactivation of specific transporters is important for fast rearrangement of the PM. In eukaryotic cells, endocytosis plays a role in transporter inactivation, which is triggered by ubiquitination of these transporters. The Nedd4 family E3 ubiquitin ligase is responsible for ubiquitination of the PM transporters and requires that a series of α-arrestin proteins are targeted to these transporters. The mechanism by which an α-arrestin recognizes its cognate transporters in response to environmental signals is of intense scientific interest. Excess substrates or signal transduction pathways are known to initiate recognition of transporters by α-arrestins. Here, we identified an endocytic-sorting signal in the monocarboxylate transporter Jen1 from yeast (Saccharomyces cerevisiae), whose endocytic degradation depends on the Snf1-glucose signaling pathway. We found that the C-terminal 20-amino acid-long region of Jen1 contains an amino acid sequence required for association of Jen1 to the α-arrestin Rod1, as well as lysine residues important for glucose-induced Jen1 ubiquitination. Notably, fusion of this region to the methionine permease, Mup1, whose endocytosis is normally induced by excess methionine, was sufficient for Mup1 to undergo glucose-induced, Rod1-mediated endocytosis. Taken together, our results demonstrate that the Jen1 C-terminal region acts as a glucose–responding degron for α-arrestin-mediated endocytic degradation of Jen1.
      PubDate: 2018-07-13T00:10:27-07:00
      DOI: 10.1074/jbc.RA117.001062
      Issue No: Vol. 293, No. 28 (2018)
       
  • Arginine methylation of translocated in liposarcoma (TLS) inhibits its
           binding to long noncoding RNA, abrogating TLS-mediated repression of
           CBP/p300 activity [RNA]
    • Authors: Wei Cui; Ryoma Yoneda, Naomi Ueda, Riki Kurokawa
      Pages: 10937 - 10948
      Abstract: Translocated in liposarcoma (TLS) is an RNA-binding protein and a transcription-regulatory sensor of DNA damage. TLS binds promoter-associated noncoding RNA (pncRNA) and inhibits histone acetyltransferase (HAT) activity of CREB-binding protein (CBP)/E1A-binding protein P300 (p300) on the cyclin D1 (CCND1) gene. Although post-translational modifications of TLS, such as arginine methylation, are known to regulate TLS's nucleocytoplasmic shuttling and assembly in stress granules, its interactions with RNAs remain poorly characterized. Herein, using various biochemical assays, we confirmed the earlier observations that TLS is methylated by protein arginine methyltransferase 1 (PRMT1) in vitro. The arginine methylation of TLS disrupted binding to pncRNA and also prevented binding of TLS to and inhibition of CBP/p300. This result indicated that arginine methylation of TLS abrogates both binding to pncRNA and TLS-mediated inhibition of CBP/p300 HAT activities. We also report that an arginine residue within the Arg–Gly–Gly domain of TLS, Arg-476, serves as the major determinant for binding to pncRNA. Either methylation or mutation of Arg-476 of TLS significantly decreased pncRNA binding and thereby prevented a pncRNA-induced allosteric alteration in TLS that is required for its interaction with CBP/p300. Moreover, unlike WT TLS, an R476A TLS mutant did not inhibit CCND1 promoter activity in luciferase reporter assays. Taken together, we propose the hypothesis that arginine methylation of TLS regulates both TLS–nucleic acid and TLS–protein interactions and thereby participates in transcriptional regulation.
      PubDate: 2018-07-13T00:10:27-07:00
      DOI: 10.1074/jbc.RA117.000598
      Issue No: Vol. 293, No. 28 (2018)
       
  • Sequence diversity of tubulin isotypes in regulation of the mitochondrial
           voltage-dependent anion channel [Molecular Biophysics]
    • Authors: Tatiana K. Rostovtseva; Philip A. Gurnev, David P. Hoogerheide, Amandine Rovini, Minhajuddin Sirajuddin, Sergey M. Bezrukov
      Pages: 10949 - 10962
      Abstract: The microtubule protein tubulin is a heterodimer comprising α/β subunits, in which each subunit features multiple isotypes in vertebrates. For example, seven α-tubulin and eight β-tubulin isotypes in the human tubulin gene family vary mostly in the length and primary sequence of the disordered anionic carboxyl-terminal tails (CTTs). The biological reason for such sequence diversity remains a topic of vigorous enquiry. Here, we demonstrate that it may be a key feature of tubulin's role in regulation of the permeability of the mitochondrial outer membrane voltage-dependent anion channel (VDAC). Using recombinant yeast α/β-tubulin constructs with α-CTTs, β-CTTs, or both from various human tubulin isotypes, we probed their interactions with VDAC reconstituted into planar lipid bilayers. A comparative study of the blockage kinetics revealed that either α-CTTs or β-CTTs block the VDAC pore and that the efficiency of blockage by individual CTTs spans 2 orders of magnitude, depending on the CTT isotype. β-Tubulin constructs, notably β3, blocked VDAC most effectively. We quantitatively described these experimental results using a physical model that accounted only for the number and distribution of charges in the CTT, and not for the interactions between specific residues on the CTT and VDAC pore. Based on these results, we speculate that the effectiveness of VDAC regulation by tubulin depends on the predominant tubulin isotype in a cell. Consequently, the fluxes of ATP/ADP through the channel could vary significantly, depending on the isotype, thus suggesting an intriguing link between VDAC regulation and the diversity of tubulin isotypes present in vertebrates.
      PubDate: 2018-07-13T00:10:27-07:00
      DOI: 10.1074/jbc.RA117.001569
      Issue No: Vol. 293, No. 28 (2018)
       
  • A heterodimer formed by bone morphogenetic protein 9 (BMP9) and BMP10
           provides most BMP biological activity in plasma [Molecular Bases of
           Disease]
    • Authors: Emmanuelle Tillet; Marie Ouarne, Agnes Desroches–Castan, Christine Mallet, Mariela Subileau, Robin Didier, Anna Lioutsko, Guillaume Belthier, Jean–Jacques Feige, Sabine Bailly
      Pages: 10963 - 10974
      Abstract: Bone morphogenetic protein 9 (BMP9) and BMP10 are the two high-affinity ligands for the endothelial receptor activin receptor-like kinase 1 (ALK1) and are key regulators of vascular remodeling. They are both present in the blood, but their respective biological activities are still a matter of debate. The aim of the present work was to characterize their circulating forms to better understand how their activities are regulated in vivo. First, by cotransfecting BMP9 and BMP10, we found that both can form a disulfide-bonded heterodimer in vitro and that this heterodimer is functional on endothelial cells via ALK1. Next, we developed an ELISA that could specifically recognize the BMP9–BMP10 heterodimer and which indicated its presence in both human and mouse plasma. In addition to using available Bmp9-KO mice, we generated a conditional Bmp10-KO mouse strain. The plasma from Bmp10-KO mice, similarly to that of Bmp9-KO mice, completely lacked the ability to activate ALK1-transfected 3T3 cells or phospho-Smad1–5 on endothelial cells, indicating that the circulating BMP activity is mostly due to the BMP9–BMP10 heterodimeric form. This result was confirmed in human plasma that had undergone affinity chromatography to remove BMP9 homodimer. Finally, we provide evidence that hepatic stellate cells in the liver could be the source of the BMP9–BMP10 heterodimer. Together, our findings demonstrate that BMP9 and BMP10 can heterodimerize and that this heterodimer is responsible for most of the biological BMP activity found in plasma.
      PubDate: 2018-07-13T00:10:27-07:00
      DOI: 10.1074/jbc.RA118.002968
      Issue No: Vol. 293, No. 28 (2018)
       
  • Interleukin-6-mediated trans-signaling inhibits transforming growth
           factor-{beta} signaling in trabecular meshwork cells [Molecular Bases of
           Disease]
    • Authors: Miyuki Inoue-Mochita; Toshihiro Inoue, Sachi Kojima, Akiko Futakuchi, Tomokazu Fujimoto, Saori Sato-Ohira, Utako Tsutsumi, Hidenobu Tanihara
      Pages: 10975 - 10984
      Abstract: Glaucoma is one of the major causes of blindness, and transforming growth factor-β2 (TGF-β2) has been found to be elevated in the aqueous humor of eyes with primary open-angle glaucoma (POAG). TGF-β2 in aqueous humor causes the glaucoma-related fibrosis of human trabecular meshwork (HTM), suggesting an important role of TGF-β in POAG pathogenesis. Here, we sought to elucidate the effects of IL-6 trans-signaling on TGF-β signaling in HTM cells. Using a multiplex immunoassay, POAG patients decreased IL-6 levels and increased soluble IL-6 receptor (sIL-6R) levels compared with the controls. In in vitro experiments, we observed that the IL-6 level was increased in the conditioned medium of HTM cells after TGF-β2 stimulation. To elucidate the relationship between TGF-β2 and IL-6 in HTM cells, we conducted Western blotting and immunohistochemical analyses, and we noted that the combination of IL-6 and sIL-6R (IL6/sIL-6R) suppressed TGF-β–induced up-regulation of α-smooth muscle actin in HTM cells, whereas IL-6 alone did not. This suggests that trans-signaling, not classic signaling, of IL-6 suppresses TGF-β–induced fibrosis of HTM. IL6/sIL-6R also suppressed TGF-β–mediated activation of myosin light chain 2 (MLC2), Smad2, and p38. Of note, these inhibitory effects of IL6/sIL-6R on TGF-β were partly reduced by siRNA-mediated knockdown of STAT3. Moreover, IL-6/sIL-6R partly inhibited TGF-β–induced activation of the Smad-sensitive promoter detected with luciferase reporter gene assays and up-regulation of TGFRI and TGFRII, evaluated by quantitative real-time RT-PCR. Strikingly, overexpression of TGFRI and TGFRII diminished these inhibitory effects of IL-6/sIL-6R. We conclude that of IL-6–mediated trans-signaling potently represses TGF-β signaling in HTM cells.
      PubDate: 2018-07-13T00:10:27-07:00
      DOI: 10.1074/jbc.RA118.003298
      Issue No: Vol. 293, No. 28 (2018)
       
  • Structural basis for selective inhibition of human PKG I{alpha} by the
           balanol-like compound N46 [Protein Structure and Folding]
    • Authors: Liying Qin; Banumathi Sankaran, Sahar Aminzai, Darren E. Casteel, Choel Kim
      Pages: 10985 - 10992
      Abstract: Activation of protein kinase G (PKG) Iα in nociceptive neurons induces long-term hyperexcitability that causes chronic pain. Recently, a derivative of the fungal metabolite balanol, N46, has been reported to inhibit PKG Iα with high potency and selectivity and attenuate thermal hyperalgesia and osteoarthritic pain. Here we determined co-crystal structures of the PKG Iα C-domain and cAMP-dependent protein kinase (PKA) Cα, each bound with N46, at 1.98 Å and 2.65 Å, respectively. N46 binds the active site with its external phenyl ring, specifically interacting with the glycine-rich loop and the αC helix. Phe-371 at the PKG Iα glycine-rich loop is oriented parallel to the phenyl ring of N46, forming a strong π-stacking interaction, whereas the analogous Phe-54 in PKA Cα rotates 30° and forms a weaker interaction. Structural comparison revealed that steric hindrance between the preceding Ser-53 and the propoxy group of the phenyl ring may explain the weaker interaction with PKA Cα. The analogous Gly-370 in PKG Iα, however, causes little steric hindrance with Phe-371. Moreover, Ile-406 on the αC helix forms a hydrophobic interaction with N46 whereas its counterpart in PKA, Thr-88, does not. Substituting these residues in PKG Iα with those in PKA Cα increases the IC50 values for N46, whereas replacing these residues in PKA Cα with those in PKG Iα reduces the IC50, consistent with our structural findings. In conclusion, our results explain the structural basis for N46-mediated selective inhibition of human PKG Iα and provide a starting point for structure-guided design of selective PKG Iα inhibitors.
      PubDate: 2018-07-13T00:10:27-07:00
      DOI: 10.1074/jbc.RA118.002427
      Issue No: Vol. 293, No. 28 (2018)
       
  • The stimulatory G protein Gs{alpha} is required in melanocortin 4
           receptor-expressing cells for normal energy balance, thermogenesis, and
           glucose metabolism [Signal Transduction]
    • Authors: Brandon Podyma; Hui Sun, Eric A. Wilson, Bradley Carlson, Ethan Pritikin, Oksana Gavrilova, Lee S. Weinstein, Min Chen
      Pages: 10993 - 11005
      Abstract: Central melanocortin 4 receptors (MC4Rs) stimulate energy expenditure and inhibit food intake. MC4Rs activate the G protein Gsα, but whether Gsα mediates all MC4R actions has not been established. Individuals with Albright hereditary osteodystrophy (AHO), who have heterozygous Gsα-inactivating mutations, only develop obesity when the Gsα mutation is present on the maternal allele because of tissue-specific genomic imprinting. Furthermore, evidence in mice implicates Gsα imprinting within the central nervous system (CNS) in this disorder. In this study, we examined the effects of Gsα in MC4R-expressing cells on metabolic regulation. Mice with homozygous Gsα deficiency in MC4R-expressing cells (MC4RGsKO) developed significant obesity with increased food intake and decreased energy expenditure, along with impaired insulin sensitivity and cold-induced thermogenesis. Moreover, the ability of the MC4R agonist melanotan-II (MTII) to stimulate energy expenditure and to inhibit food intake was impaired in MC4RGsKO mice. MTII failed to stimulate the secretion of the anorexigenic hormone peptide YY (PYY) from enteroendocrine L cells, a physiological response mediated by MC4R–Gsα signaling, even though baseline PYY levels were elevated in these mice. In Gsα heterozygotes, mild obesity and reduced energy expenditure were present only in mice with a Gsα deletion on the maternal allele in MC4R-expressing cells, whereas food intake was unaffected. These results demonstrate that Gsα signaling in MC4R-expressing cells is required for controlling energy balance, thermogenesis, and peripheral glucose metabolism. They further indicate that Gsα imprinting in MC4R-expressing cells contributes to obesity in Gsα knockout mice and probably in individuals with Albright hereditary osteodystrophy as well.
      PubDate: 2018-07-13T00:10:27-07:00
      DOI: 10.1074/jbc.RA118.003450
      Issue No: Vol. 293, No. 28 (2018)
       
  • Select {alpha}-arrestins control cell-surface abundance of the mammalian
           Kir2.1 potassium channel in a yeast model [Membrane Biology]
    • Authors: Natalie A. Hager; Collin J. Krasowski, Timothy D. Mackie, Alexander R. Kolb, Patrick G. Needham, Andrew A. Augustine, Alison Dempsey, Christopher Szent-Gyorgyi, Marcel P. Bruchez, Daniel J. Bain, Adam V. Kwiatkowski, Allyson F. O'Donnell, Jeffrey L. Brodsky
      Pages: 11006 - 11021
      Abstract: Protein composition at the plasma membrane is tightly regulated, with rapid protein internalization and selective targeting to the cell surface occurring in response to environmental changes. For example, ion channels are dynamically relocalized to or from the plasma membrane in response to physiological alterations, allowing cells and organisms to maintain osmotic and salt homeostasis. To identify additional factors that regulate the selective trafficking of a specific ion channel, we used a yeast model for a mammalian potassium channel, the K+ inward rectifying channel Kir2.1. Kir2.1 maintains potassium homeostasis in heart muscle cells, and Kir2.1 defects lead to human disease. By examining the ability of Kir2.1 to rescue the growth of yeast cells lacking endogenous potassium channels, we discovered that specific α-arrestins regulate Kir2.1 localization. Specifically, we found that the Ldb19/Art1, Aly1/Art6, and Aly2/Art3 α-arrestin adaptor proteins promote Kir2.1 trafficking to the cell surface, increase Kir2.1 activity at the plasma membrane, and raise intracellular potassium levels. To better quantify the intracellular and cell-surface populations of Kir2.1, we created fluorogen-activating protein fusions and for the first time used this technique to measure the cell-surface residency of a plasma membrane protein in yeast. Our experiments revealed that two α-arrestin effectors also control Kir2.1 localization. In particular, both the Rsp5 ubiquitin ligase and the protein phosphatase calcineurin facilitated the α-arrestin–mediated trafficking of Kir2.1. Together, our findings implicate α-arrestins in regulating an additional class of plasma membrane proteins and establish a new tool for dissecting the trafficking itinerary of any membrane protein in yeast.
      PubDate: 2018-07-13T00:10:27-07:00
      DOI: 10.1074/jbc.RA117.001293
      Issue No: Vol. 293, No. 28 (2018)
       
  • Coupling of Smoothened to inhibitory G proteins reduces voltage-gated K+
           currents in cardiomyocytes and prolongs cardiac action potential duration
           [Cell Biology]
    • Authors: Lan Cheng; Moza Al-Owais, Manuel L. Covarrubias, Walter J. Koch, David. R. Manning, Chris Peers, Natalia A. Riobo-Del Galdo
      Pages: 11022 - 11032
      Abstract: SMO (Smoothened), the central transducer of Hedgehog signaling, is coupled to heterotrimeric Gi proteins in many cell types, including cardiomyocytes. In this study, we report that activation of SMO with SHH (Sonic Hedgehog) or a small agonist, purmorphamine, rapidly causes a prolongation of the action potential duration that is sensitive to a SMO inhibitor. In contrast, neither of the SMO agonists prolonged the action potential in cardiomyocytes from transgenic GiCT/TTA mice, in which Gi signaling is impaired, suggesting that the effect of SMO is mediated by Gi proteins. Investigation of the mechanism underlying the change in action potential kinetics revealed that activation of SMO selectively reduces outward voltage-gated K+ repolarizing (Kv) currents in isolated cardiomyocytes and that it induces a down-regulation of membrane levels of Kv4.3 in cardiomyocytes and intact hearts from WT but not from GiCT/TTA mice. Moreover, perfusion of intact hearts with Shh or purmorphamine increased the ventricular repolarization time (QT interval) and induced ventricular arrhythmias. Our data constitute the first report that acute, noncanonical Hh signaling mediated by Gi proteins regulates K+ currents density in cardiomyocytes and sensitizes the heart to the development of ventricular arrhythmias.
      PubDate: 2018-07-13T00:10:27-07:00
      DOI: 10.1074/jbc.RA118.001989
      Issue No: Vol. 293, No. 28 (2018)
       
  • Lysophospholipids induce innate immune transdifferentiation of endothelial
           cells, resulting in prolonged endothelial activation [Gene Regulation]
    • Authors: Xinyuan Li; Luqiao Wang, Pu Fang, Yu Sun, Xiaohua Jiang, Hong Wang, Xiao-Feng Yang
      Pages: 11033 - 11045
      Abstract: Innate immune cells express danger-associated molecular pattern (DAMP) receptors, T-cell costimulation/coinhibition receptors, and major histocompatibility complex II (MHC-II). We have recently proposed that endothelial cells can serve as innate immune cells, but the molecular mechanisms involved still await discovery. Here, we investigated whether human aortic endothelial cells (HAECs) could be transdifferentiated into innate immune cells by exposing them to hyperlipidemia–up-regulated DAMP molecules, i.e. lysophospholipids. Performing RNA-seq analysis of lysophospholipid-treated HAECs, we found that lysophosphatidylcholine (LPC) and lysophosphatidylinositol (LPI) regulate largely distinct gene programs as revealed by principal component analysis. Metabolically, LPC up-regulated genes that are involved in cholesterol biosynthesis, presumably through sterol regulatory element–binding protein 2 (SREBP2). By contrast, LPI up-regulated gene transcripts critical for the metabolism of glucose, lipids, and amino acids. Of note, we found that LPC and LPI both induce adhesion molecules, cytokines, and chemokines, which are all classic markers of endothelial cell activation, in HAECs. Moreover, LPC and LPI shared the ability to transdifferentiate HAECs into innate immune cells, including induction of potent DAMP receptors, such as CD36 molecule, T-cell costimulation/coinhibition receptors, and MHC-II proteins. The induction of these innate-immunity signatures by lysophospholipids correlated with their ability to induce up-regulation of cytosolic calcium and mitochondrial reactive oxygen species. In conclusion, lysophospholipids such as LPC and LPI induce innate immune cell transdifferentiation in HAECs. The concept of prolonged endothelial activation, discovered here, is relevant for designing new strategies for managing cardiovascular diseases.
      PubDate: 2018-07-13T00:10:27-07:00
      DOI: 10.1074/jbc.RA118.002752
      Issue No: Vol. 293, No. 28 (2018)
       
  • Protein kinase A-mediated phosphorylation of serine 357 of the mouse
           prostacyclin receptor regulates its coupling to Gs-, to Gi-, and to
           Gq-coupled effector signaling. [Additions and Corrections]
    • Authors: Orlaith A. Lawler; Sinead M. Miggin, B. Therese Kinsella
      Pages: 11046 - 11046
      Abstract: VOLUME 276 (2001) PAGES 33596–33607PAGE 33605:During a recent review of this article, the authors realized that there may have been unspecified reordering of lanes in Fig. 11B and possible duplication of lanes 2 between Fig. 11, B and C. As the original data were no longer available, replicate data are provided. This correction does not affect the results or conclusions of this work. The authors wish to apologize for any inconvenience this error may have caused.jbc;293/28/11046/FU1F1FU1
      PubDate: 2018-07-13T00:10:27-07:00
      DOI: 10.1074/jbc.AAC118.004491
      Issue No: Vol. 293, No. 28 (2018)
       
  • RNA-binding proteins with basic-acidic dipeptide (BAD) domains
           self-assemble and aggregate in Alzheimer's disease [Genomics and
           Proteomics]
    • Authors: Isaac Bishof; Eric B. Dammer, Duc M. Duong, Sean R. Kundinger, Marla Gearing, James J. Lah, Allan I. Levey, Nicholas T. Seyfried
      Pages: 11047 - 11066
      Abstract: The U1 small nuclear ribonucleoprotein 70 kDa (U1-70K) and other RNA-binding proteins (RBPs) are mislocalized to cytoplasmic neurofibrillary Tau aggregates in Alzheimer's disease (AD), yet the co-aggregation mechanisms are incompletely understood. U1-70K harbors two disordered low–complexity domains (LC1 and LC2) that are necessary for aggregation in AD brain extracts. The LC1 domain contains highly repetitive basic (Arg/Lys) and acidic (Asp/Glu) residues, referred to as a basic-acidic dipeptide (BAD) domain. We report here that this domain shares many of the properties of the Gln/Asn-rich LC domains in RBPs that also aggregate in neurodegenerative disease. These properties included self-assembly into oligomers and localization to nuclear granules. Co-immunoprecipitations of recombinant U1-70K and deletions lacking the LC domain(s) followed by quantitative proteomic analyses were used to resolve functional classes of U1-70K-interacting proteins that depend on the BAD domain for their interaction. Within this interaction network, we identified a class of RBPs with BAD domains nearly identical to that found in U1-70K. Two members of this class, LUC7L3 and RBM25, required their respective BAD domains for reciprocal interactions with U1-70K and nuclear granule localization. Strikingly, a significant proportion of RBPs with BAD domains had elevated insolubility in the AD brain proteome. Furthermore, we show that the BAD domain of U1-70K can interact with Tau from AD brains but not from other tauopathies. These findings highlight a mechanistic role for BAD domains in stabilizing RBP interactions and in potentially mediating co-aggregation with the pathological AD–specific Tau isoforms.
      PubDate: 2018-07-13T00:10:27-07:00
      DOI: 10.1074/jbc.RA118.001747
      Issue No: Vol. 293, No. 28 (2018)
       
  • JAK1-mediated Sirt1 phosphorylation functions as a negative feedback of
           the JAK1-STAT3 pathway [Signal Transduction]
    • Authors: Wenhui Wang; Fei Li, Yuanming Xu, Juncheng Wei, Yana Zhang, Heeyoung Yang, Beixue Gao, Guohua Yu, Deyu Fang
      Pages: 11067 - 11075
      Abstract: The type III NAD–dependent histone deacetylase Sirt1 plays important roles in a variety of pathobiological functions through targeting either the acetylated histones or transcription factors. However, the molecular mechanisms underlying how the Sirt1 functions are regulated remain vague. Herein we identified that the Janus kinase 1 (JAK1) interacts with Sirt1 and catalyzes its phosphorylation at the tyrosine residues of 280 and 301, both of which are highly conserved and located in the histone deacetylase catalytic domain of Sirt1. IL-6 stimulation enhanced Sirt1 interaction with JAK1 and JAK1-mediated Sirt1 phosphorylation. Interestingly, JAK1-mediated Sirt1 phosphorylation did not alter Sirt1 deacetylase catalytic activity, but instead it is required for Sirt1 interaction with the downstream transcription factor STAT3. JAK1-mediated phosphorylation enhanced Sirt1 suppression of STAT3 acetylation and transcriptional activity. As a consequence, Sirt1 activation attenuates IL-6 activity in protecting cancer cells from chemotherapeutic drug–induced apoptosis. Our studies identify JAK1 as a previously unappreciated tyrosine kinase of Sirt1 and reveal a novel negative feedback of the JAK1-STAT3 pathway.
      PubDate: 2018-07-13T00:10:27-07:00
      DOI: 10.1074/jbc.RA117.001387
      Issue No: Vol. 293, No. 28 (2018)
       
  • Decline in arylsulfatase B expression increases EGFR expression by
           inhibiting the protein-tyrosine phosphatase SHP2 and activating JNK in
           prostate cells [Signal Transduction]
    • Authors: Sumit Bhattacharyya; Leo Feferman, Xiaorui Han, Yilan Ouyang, Fuming Zhang, Robert J. Linhardt, Joanne K. Tobacman
      Pages: 11076 - 11087
      Abstract: Epidermal growth factor receptor (EGFR) has a crucial role in cell differentiation and proliferation and cancer, and its expression appears to be up-regulated when arylsulfatase B (ARSB or GalNAc-4-sulfatase) is reduced. ARSB removes 4-sulfate groups from the nonreducing end of dermatan sulfate and chondroitin 4-sulfate (C4S), and its decreased expression has previously been reported to inhibit the activity of the ubiquitous protein-tyrosine phosphatase, nonreceptor type 11 (SHP2 or PTPN11). However, the mechanism by which decline in ARSB leads to decline in SHP2 activity is unclear. Here, we show that SHP2 binds preferentially C4S, rather than chondroitin 6-sulfate, and confirm that SHP2 activity declines when ARSB is silenced. The reduction in ARSB activity, and the resultant increase in C4S, increased the expression of EGFR (Her1/ErbB1) in human prostate stem and epithelial cells. The increased expression of EGFR occurred after 1) the decline in SHP2 activity, 2) enhanced c-Jun N-terminal kinase (JNK) activity, 3) increased nuclear DNA binding by c-Jun and c-Fos, and 4) EGFR promoter activation. In response to exogenous EGF, there was increased bromodeoxyuridine incorporation, consistent with enhanced cell proliferation. These findings indicated that ARSB and chondroitin 4-sulfation affect the activation of an important dual phosphorylation threonine–tyrosine kinase and the mRNA expression of a critical tyrosine kinase receptor in prostate cells. Restoration of ARSB activity with the associated reduction in C4S may provide a new therapeutic approach for managing malignancies in which EGFR-mediated tyrosine kinase signaling pathways are active.
      PubDate: 2018-07-13T00:10:27-07:00
      DOI: 10.1074/jbc.RA117.001244
      Issue No: Vol. 293, No. 28 (2018)
       
  • Structural basis for ADP-dependent glucokinase inhibition by
           8-bromo-substituted adenosine nucleotide [Protein Structure and Folding]
    • Authors: Przemysław Grudnik; Marcin M. Kamiłski, Krzysztof P. Rembacz, Katarzyna Kuśka, Mariusz Madeȷ, Jan Potempa, Macieȷ Dawidowski, Grzegorz Dubin
      Pages: 11088 - 11099
      Abstract: In higher eukaryotes, several ATP-utilizing enzymes known as hexokinases activate glucose in the glycolysis pathway by phosphorylation to glucose 6-phosphate. In contrast to canonical hexokinases, which use ATP, ADP-dependent glucokinase (ADPGK) catalyzes noncanonical phosphorylation of glucose to glucose 6-phosphate using ADP as a phosphate donor. Initially discovered in Archaea, the human homolog of ADPGK was described only recently. ADPGK's involvement in modified bioenergetics of activated T cells has been postulated, and elevated ADPGK expression has been reported in various cancer tissues. However, the physiological role of ADPGK is still poorly understood, and effective ADPGK inhibitors still await discovery. Here, we show that 8-bromo–substituted adenosine nucleotide inhibits human ADPGK. By solving the crystal structure of archaeal ADPGK in complex with 8-bromoadenosine phosphate (8-Br-AMP) at 1.81 Å resolution, we identified the mechanism of inhibition. We observed that 8-Br-AMP is a competitive inhibitor of ADPGK and that the bromine substitution induces marked structural changes within the protein's active site by engaging crucial catalytic residues. The results obtained using the Jurkat model of activated human T cells suggest its moderate activity in a cellular setting. We propose that our structural insights provide a critical basis for rational development of novel ADPGK inhibitors.
      PubDate: 2018-07-13T00:10:27-07:00
      DOI: 10.1074/jbc.RA117.001562
      Issue No: Vol. 293, No. 28 (2018)
       
  • Impact of tobacco-specific nitrosamine-derived DNA adducts on the
           efficiency and fidelity of DNA replication in human cells [DNA and
           Chromosomes]
    • Authors: Hua Du; Jiapeng Leng, Pengcheng Wang, Lin Li, Yinsheng Wang
      Pages: 11100 - 11108
      Abstract: The tobacco-derived nitrosamines 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) and N′-nitrosonornicotine (NNN) are known human carcinogens. Following metabolic activation, NNK and NNN can induce a number of DNA lesions, including several 4-(3-pyridyl)-4-oxobut-1-yl (POB) adducts. However, it remains unclear to what extent these lesions affect the efficiency and accuracy of DNA replication and how their replicative bypass is influenced by translesion synthesis (TLS) DNA polymerases. In this study, we investigated the effects of three stable POB DNA adducts (O2-POB-dT, O4-POB-dT, and O6-POB-dG) on the efficiency and fidelity of DNA replication in HEK293T human cells. We found that, when situated in a double-stranded plasmid, O2-POB-dT and O4-POB-dT moderately blocked DNA replication and induced exclusively T→A (∼14.9%) and T→C (∼35.2%) mutations, respectively. On the other hand, O6-POB-dG slightly impeded DNA replication, and this lesion elicited primarily the G→A transition (∼75%) together with a low frequency of the G→T transversion (∼3%). By conducting replication studies in isogenic cells in which specific TLS DNA polymerases (Pols) were deleted by CRISPR-Cas9 genome editing, we observed that multiple TLS Pols, especially Pol η and Pol ζ, are involved in bypassing these lesions. Our findings reveal the cytotoxic and mutagenic properties of specific POB DNA adducts and unravel the roles of several TLS polymerases in the replicative bypass of these adducts in human cells. Together, these results provide important new knowledge about the biological consequences of POB adducts.
      PubDate: 2018-07-13T00:10:27-07:00
      DOI: 10.1074/jbc.RA118.003477
      Issue No: Vol. 293, No. 28 (2018)
       
  • The transcription factor Vezf1 represses the expression of the
           antiangiogenic factor Cited2 in endothelial cells [DNA and Chromosomes]
    • Authors: Lama AlAbdi; Ming He, Qianyi Yang, Allison B. Norvil, Humaira Gowher
      Pages: 11109 - 11118
      Abstract: Formation of the vasculature by angiogenesis is critical for proper development, but angiogenesis also contributes to the pathogenesis of various disorders, including cancer and cardiovascular diseases. Vascular endothelial zinc finger 1 (Vezf1), is a Krüppel-like zinc finger protein that plays a vital role in vascular development. However, the mechanism by which Vezf1 regulates this process is not fully understood. Here, we show that Vezf1−/− mouse embryonic stem cells (ESC) have significantly increased expression of a stem cell factor, Cbp/p300-interacting transactivator 2 (Cited2). Compared with WT ESCs, Vezf1−/− ESCs inefficiently differentiated into endothelial cells (ECs), which exhibited defects in the tube-formation assay. These defects were due to reduced activation of EC-specific genes concomitant with lower enrichment of histone 3 acetylation at Lys27 (H3K27) at their promoters. We hypothesized that overexpression of Cited2 in Vezf1−/− cells sequesters P300/CBP away from the promoters of proangiogenic genes and thereby contributes to defective angiogenesis in these cells. This idea was supported by the observation that shRNA-mediated depletion of Cited2 significantly reduces the angiogenic defects in the Vezf1−/− ECs. In contrast to previous studies that have focused on the role of Vezf1 as a transcriptional activator of proangiogenic genes, our findings have revealed a role for Vezf1 in modulating the expression of the antiangiogenic factor Cited2. Vezf1 previously has been characterized as an insulator protein, and our results now provide insights into the mechanism, indicating that Vezf1 can block inappropriate, nonspecific interactions of promoters with cis-located enhancers, preventing aberrant promoter activation.
      PubDate: 2018-07-13T00:10:27-07:00
      DOI: 10.1074/jbc.RA118.002911
      Issue No: Vol. 293, No. 28 (2018)
       
  • Protein kinase C{alpha} drives fibroblast activation and kidney fibrosis
           by stimulating autophagic flux [Molecular Bases of Disease]
    • Authors: Xian Xue; Jiafa Ren, Xiaoli Sun, Yuan Gui, Ye Feng, Bingyan Shu, Wei Wei, Qingmiao Lu, Yan Liang, Weichun He, Junwei Yang, Chunsun Dai
      Pages: 11119 - 11130
      Abstract: Kidney fibrosis is a histological hallmark of chronic kidney disease and arises in large part through extracellular matrix deposition by activated fibroblasts. The signaling protein complex mTOR complex 2 (mTORC2) plays a critical role in fibroblast activation and kidney fibrosis. Protein kinase Cα (PKCα) is one of the major sub-pathways of mTORC2, but its role in fibroblast activation and kidney fibrosis remains to be determined. Here, we found that transforming growth factor β1 (TGFβ1) activates PKCα signaling in cultured NRK-49F cells in a time-dependent manner. Blocking PKCα signaling with the chemical inhibitor Go6976 or by transfection with PKCα siRNA largely reduced expression of the autophagy-associated protein lysosomal-associated membrane protein 2 (LAMP2) and also inhibited autophagosome–lysosome fusion and autophagic flux in the cells. Similarly to chloroquine, Go6976 treatment and PKCα siRNA transfection also markedly inhibited TGFβ1-induced fibroblast activation. In murine fibrotic kidneys with unilateral ureteral obstruction (UUO) nephropathy, PKCα signaling is activated in the interstitial myofibroblasts. Go6976 administration largely blocked autophagic flux in fibroblasts in the fibrotic kidneys and attenuated the UUO nephropathy. Together, our findings suggest that blocking PKCα activity may retard autophagic flux and thereby prevent fibroblast activation and kidney fibrosis.
      PubDate: 2018-07-13T00:10:27-07:00
      DOI: 10.1074/jbc.RA118.002191
      Issue No: Vol. 293, No. 28 (2018)
       
  • Restoration of hydroxyindole O-methyltransferase levels in human cancer
           cells induces a tryptophan-metabolic switch and attenuates cancer
           progression [Metabolism]
    • Authors: Hua-Ling Chen; Chao-Yun Yuan, Huei-Hsuan Cheng, Tzu-Ching Chang, Shau-Ku Huang, Cheng-Chin Kuo, Kenneth K. Wu
      Pages: 11131 - 11142
      Abstract: 5-Methoxytryptophan (5-MTP) is a tryptophan metabolite with recently discovered anti-inflammatory and tumor-suppressing activities. Its synthesis is catalyzed by a hydroxyindole O-methyltransferase (HIOMT)-like enzyme. However, the exact identity of this HIOMT in human cells remains unclear. Human HIOMT exists in several alternatively spliced isoforms, and we hypothesized that 5-MTP–producing HIOMT is a distinct isoform. Here, we show that human fibroblasts and cancer cells express the HIOMT298 isoform as contrasted with the expression of the HIOMT345 isoform in pineal cells. Sequencing analysis of the cloned isoforms revealed that HIOMT298 is identical to the sequence of a previously reported truncated HIOMT isoform. Of note, HIOMT298 expression was reduced in cancer cells and tissues. Stable transfection of A549 cancer cells with HIOMT298 restored HIOMT expression to normal levels, accompanied by 5-MTP production. Furthermore, HIOMT298 transfection caused a tryptophan-metabolic switch from serotonin to 5-MTP production. To determine the in vivo relevance of this alteration, we compared growth and lung metastasis of HIOMT298-transfected A549 cells with those of vector- or untransfected A549 cells as controls in a murine xenograft model. Of note, the HIOMT298-transfected A549 cells exhibited slower growth and lower metastasis than the controls. Our findings provide insight into the crucial role of HIOMT298 in 5-MTP production in cells and in inhibiting cancer progression and highlight the potential therapeutic value of 5-MTP for managing cancer.
      PubDate: 2018-07-13T00:10:27-07:00
      DOI: 10.1074/jbc.RA117.000597
      Issue No: Vol. 293, No. 28 (2018)
       
  • Interleukin-6 promotes pancreatic cancer cell migration by rapidly
           activating the small GTPase CDC42 [Cell Biology]
    • Authors: Gina L. Razidlo; Kevin M. Burton, Mark A. McNiven
      Pages: 11143 - 11153
      Abstract: Inflammation is a major driver of tumor progression and metastasis, although the mechanisms by which proinflammatory cytokines drive metastatic invasion are unknown. Interleukin-6 (IL-6) is a potent proinflammatory cytokine that is elevated in individuals with pancreatic cancer (PDAC), is required for PDAC progression in mice, and increases tumor cell invasion in vitro. Here, we provide insights into the mechanisms by which IL-6 activates tumor cell invasion. We found that IL-6 stimulation rapidly and robustly activates the small GTPase cell division cycle 42 (CDC42) in human PDAC cells and promotes the formation of premigratory filopodia. The CDC42 activation was required for IL-6–induced invasion as blocking CDC42 activity rendered the cells insensitive to IL-6's proinvasive effects. Loss of Janus kinase 2 (JAK2) or signal transducer and activator of transcription 3 (STAT3) prevented IL-6–mediated CDC42 activation, indicating that IL-6 activates CDC42 through both JAK2 and STAT3. However, the rapid activation of CDC42 suggested that this activation may be distinct from canonical STAT3-mediated transcriptional activation. Importantly, we observed an interaction between STAT3 and IQ motif–containing GTPase-activating protein 1 (IQGAP1), a scaffolding platform that binds CDC42. STAT3 colocalized with CDC42 and IQGAP1 at the plasma membrane, suggesting cross-talk between IL-6–mediated STAT3 signaling and CDC42 activation. These results suggest that IL-6 promotes metastatic invasion, at least partially, through CDC42 and that, along with its pleiotropic effects on tumor growth and progression, IL-6 signaling also activates proinvasive GTPase signaling, priming tumor cells for metastatic invasion.
      PubDate: 2018-07-13T00:10:27-07:00
      DOI: 10.1074/jbc.RA118.003276
      Issue No: Vol. 293, No. 28 (2018)
       
  • Discovery of a new Pro-Pro endopeptidase, PPEP-2, provides mechanistic
           insights into the differences in substrate specificity within the PPEP
           family [Enzymology]
    • Authors: Oleg I. Klychnikov; Tatiana M. Shamorkina, Stephen D. Weeks, Hans C. van Leeuwen, Jeroen Corver, Jan W. Drijfhout, Peter A. van Veelen, Nikolai N. Sluchanko, Sergei V. Strelkov, Paul J. Hensbergen
      Pages: 11154 - 11165
      Abstract: Pro-Pro endopeptidases (PPEPs) belong to a recently discovered family of proteases capable of hydrolyzing a Pro–Pro bond. The first member from the bacterial pathogen Clostridium difficile (PPEP-1) cleaves two C. difficile cell-surface proteins involved in adhesion, one of which is encoded by the gene adjacent to the ppep-1 gene. However, related PPEPs may exist in other bacteria and may shed light on substrate specificity in this enzyme family. Here, we report on the homolog of PPEP-1 in Paenibacillus alvei, which we denoted PPEP-2. We found that PPEP-2 is a secreted metalloprotease, which likewise cleaved a cell-surface protein encoded by an adjacent gene. However, the cleavage motif of PPEP-2, PLP↓PVP, is distinct from that of PPEP-1 (VNP↓PVP). As a result, an optimal substrate peptide for PPEP-2 was not cleaved by PPEP-1 and vice versa. To gain insight into the specificity mechanism of PPEP-2, we determined its crystal structure at 1.75 Å resolution and further confirmed the structure in solution using small-angle X-ray scattering (SAXS). We show that a four-amino-acid loop, which is distinct in PPEP-1 and -2 (GGST in PPEP-1 and SERV in PPEP-2), plays a crucial role in substrate specificity. A PPEP-2 variant, in which the four loop residues had been swapped for those from PPEP-1, displayed a shift in substrate specificity toward PPEP-1 substrates. Our results provide detailed insights into the PPEP-2 structure and the structural determinants of substrate specificity in this new family of PPEP proteases.
      PubDate: 2018-07-13T00:10:27-07:00
      DOI: 10.1074/jbc.RA118.003244
      Issue No: Vol. 293, No. 28 (2018)
       
  • ATP-binding cassette member B5 (ABCB5) promotes tumor cell invasiveness in
           human colorectal cancer [Cell Biology]
    • Authors: Qin Guo; Tanja Grimmig, Gabriel Gonzalez, Anita Giobbie-Hurder, Gretchen Berg, Nolan Carr, Brian J. Wilson, Pallavi Banerjee, Jie Ma, Jason S. Gold, Bisweswar Nandi, Qin Huang, Ana Maria Waaga-Gasser, Christine G. Lian, George F. Murphy, Markus H. Frank, Martin Gasser, Natasha Y. Frank
      Pages: 11166 - 11178
      Abstract: ABC member B5 (ABCB5) mediates multidrug resistance (MDR) in diverse malignancies and confers clinically relevant 5-fluorouracil resistance to CD133-expressing cancer stem cells in human colorectal cancer (CRC). Because of its recently identified roles in normal stem cell maintenance, we hypothesized that ABCB5 might also serve MDR-independent functions in CRC. Here, in a prospective clinical study of 142 CRC patients, we found that ABCB5 mRNA transcripts previously reported not to be significantly expressed in healthy peripheral blood mononuclear cells are significantly enriched in patient peripheral blood specimens compared with non-CRC controls and correlate with CRC disease progression. In human-to-mouse CRC tumor xenotransplantation models that exhibited circulating tumor mRNA, we observed that cancer-specific ABCB5 knockdown significantly reduced detection of these transcripts, suggesting that the knockdown inhibited tumor invasiveness. Mechanistically, this effect was associated with inhibition of expression and downstream signaling of AXL receptor tyrosine kinase (AXL), a proinvasive molecule herein shown to be produced by ABCB5-positive CRC cells. Importantly, rescue of AXL expression in ABCB5-knockdown CRC tumor cells restored tumor-specific transcript detection in the peripheral blood of xenograft recipients, indicating that ABCB5 regulates CRC invasiveness, at least in part, by enhancing AXL signaling. Our results implicate ABCB5 as a critical determinant of CRC invasiveness and suggest that ABCB5 blockade might represent a strategy in CRC therapy, even independently of ABCB5's function as an MDR mediator.
      PubDate: 2018-07-13T00:10:27-07:00
      DOI: 10.1074/jbc.RA118.003187
      Issue No: Vol. 293, No. 28 (2018)
       
  • Striatin-1 is a B subunit of protein phosphatase PP2A that regulates
           dendritic arborization and spine development in striatal neurons [Signal
           Transduction]
    • Authors: Daniel Li; Veronica Musante, Wenliang Zhou, Marina R. Picciotto, Angus C. Nairn
      Pages: 11179 - 11194
      Abstract: Striatin-1, a subunit of the serine/threonine phosphatase PP2A, is preferentially expressed in neurons in the striatum. As a member of the striatin family of B subunits, striatin-1 is a core component together with PP2A of a multiprotein complex called STRIPAK, the striatin-interacting phosphatase and kinase complex. Little is known about the function of striatin-1 or the STRIPAK complex in the mammalian striatum. Here, we identify a selective role for striatin-1 in striatal neuron maturation. Using a small hairpin RNA (shRNA) knockdown approach in primary striatal neuronal cultures, we determined that reduced expression of striatin-1 results in increased dendritic complexity and an increased density of dendritic spines, classified as stubby spines. The dendritic phenotype was rescued by co-expression of a striatin-1 mutant construct insensitive to the knockdown shRNA but was not rescued by co-expression of PP2A- or Mob3-binding deficient striatin-1 constructs. Reduction of striatin-1 did not result in deficits in neuronal connectivity in this knockdown model, as we observed no abnormalities in synapse formation or in spontaneous excitatory postsynaptic currents. Thus, this study suggests that striatin-1 is a regulator of neuronal development in striatal neurons.
      PubDate: 2018-07-13T00:10:27-07:00
      DOI: 10.1074/jbc.RA117.001519
      Issue No: Vol. 293, No. 28 (2018)
       
  • Effects of phosphatidylcholine membrane fluidity on the conformation and
           aggregation of N-terminally acetylated {alpha}-synuclein [Lipids]
    • Authors: Emma I. O'Leary; Zhiping Jiang, Marie-Paule Strub, Jennifer C. Lee
      Pages: 11195 - 11205
      Abstract: Membrane association of α-synuclein (α-syn), a neuronal protein associated with Parkinson's disease (PD), is involved in α-syn function and pathology. Most previous studies on α-syn–membrane interactions have not used the physiologically relevant N-terminally acetylated (N-acetyl) α-syn form nor the most naturally abundant cellular lipid, i.e. phosphatidylcholine (PC). Here, we report on how PC membrane fluidity affects the conformation and aggregation propensity of N-acetyl α-syn. It is well established that upon membrane binding, α-syn adopts an α-helical structure. Using CD spectroscopy, we show that N-acetyl α-syn transitions from α-helical to disordered at the lipid melting temperature (Tm). We found that this fluidity sensing is a robust characteristic, unaffected by acyl chain length (Tm = 34–55 °C) and preserved in its homologs β- and γ-syn. Interestingly, both N-acetyl α-syn membrane binding and amyloid formation trended with lipid order (1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC)> 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC)/sphingomyelin/cholesterol (2:2:1) ≥ DOPC), with gel-phase vesicles shortening aggregation kinetics and promoting fibril formation compared to fluid membranes. Furthermore, we found that acetylation enhances binding to PC micelles and small unilamellar vesicles with high curvature (r ∼16–20 nm) and that DPPC binding is reduced in the presence of cholesterol. These results confirmed that the exposure of hydrocarbon chains (i.e. packing defects) is essential for binding to zwitterionic gel membranes. Collectively, our in vitro results suggest that N-acetyl α-syn localizes to highly curved, ordered membranes inside a cell. We propose that age-related changes in membrane fluidity can promote the formation of amyloid fibrils, insoluble materials associated with PD.
      PubDate: 2018-07-13T00:10:27-07:00
      DOI: 10.1074/jbc.RA118.002780
      Issue No: Vol. 293, No. 28 (2018)
       
  • Phosphoinositide binding by the PH domain in ceramide transfer protein
           (CERT) is inhibited by hyperphosphorylation of an adjacent serine-repeat
           motif [Protein Structure and Folding]
    • Authors: Toshihiko Sugiki; Daichi Egawa, Keigo Kumagai, Chojiro Kojima, Toshimichi Fujiwara, Koh Takeuchi, Ichio Shimada, Kentaro Hanada, Hideo Takahashi
      Pages: 11206 - 11217
      Abstract: Sphingolipids such as ceramide are important constituents of cell membranes. The ceramide transfer protein (CERT) moves ceramide from the endoplasmic reticulum to the Golgi apparatus in a nonvesicular manner. Hyperphosphorylation of the serine-repeat motif (SRM) adjacent to the pleckstrin homology (PH) domain of CERT down-regulates the inter-organelle ceramide transport function of CERT. However, the mechanistic details of this down-regulation remain elusive. Using solution NMR and binding assays, we herein show that a hyperphosphorylation-mimetic CERT variant in which 10 serine/threonine residues of SRM had been replaced with glutamate residues (the 10E variant) displays an intramolecular interaction between SRM and positively charged regions of the PH domain, which are involved in the binding of this domain to phosphatidylinositol 4-monophosphate (PI4P). Of note, the binding of the PH domain to PI4P-embedded membranes was attenuated by the SRM 10E substitutions in cell-free assays. Moreover, the 10E substitutions reduced the Golgi-targeting activity of the PH-SRM construct in living cells. These results indicate that hyperphosphorylated SRM directly interacts with the surface of the PH domain in an intramolecular manner, thereby decreasing the PI4P-binding activity of the PH domain. In light of these findings, we propose that the hyperphosphorylation of SRM may trigger the dissociation of CERT from the Golgi apparatus, resulting in a functionally less active conformation of CERT.
      PubDate: 2018-07-13T00:10:27-07:00
      DOI: 10.1074/jbc.RA118.002465
      Issue No: Vol. 293, No. 28 (2018)
       
  • Properly folded and functional PorB from Neisseria gonorrhoeae inhibits
           dendritic cell stimulation of CD4+ T cell proliferation [Immunology]
    • Authors: Weiyan Zhu; Joshua Tomberg, Kayla J. Knilans, James E. Anderson, Karen P. McKinnon, Gregory D. Sempowski, Robert A. Nicholas, Joseph A. Duncan
      Pages: 11218 - 11229
      Abstract: Neisseria gonorrhoeae is an exclusive human pathogen that evades the host immune system through multiple mechanisms. We have shown that N. gonorrhoeae suppresses the capacity of antigen-presenting cells to induce CD4+ T cell proliferation. In this study, we sought to determine the gonococcal factors involved in this adaptive immune suppression. We show that suppression of the capacity of antigen-pulsed dendritic cells to induce T cell proliferation is recapitulated by administration of a high-molecular-weight fraction of conditioned medium from N. gonorrhoeae cultures, which includes outer membrane vesicles that are shed during growth of the bacteria. N. gonorrhoeae PorB is the most abundant protein in N. gonorrhoeae–derived vesicles, and treatment of dendritic cells with purified recombinant PorB inhibited the capacity of the cells to stimulate T cell proliferation. This immunosuppressive feature of purified PorB depended on proper folding of the protein. PorB from N. gonorrhoeae, as well as other Neisseria species and other Gram-negative bacterial species, are known to activate host Toll-like receptor 2 (TLR2) signaling. Published studies have demonstrated that purified Neisseria PorB forms proteinacious nanoparticles, termed proteosomes, when detergent micelles are removed. Unlike folded, detergent-solubilized PorB, PorB proteosomes stimulate immune responses. We now demonstrate that the formation of PorB proteosomes from structurally intact PorB eliminates the immunosuppressive property of the protein while enhancing TLR2 stimulation. These findings suggest that gonococcal PorB present in shed outer membrane vesicles plays a role in suppression of adaptive immune responses to this immune-evasive pathogen.
      PubDate: 2018-07-13T00:10:27-07:00
      DOI: 10.1074/jbc.RA117.001209
      Issue No: Vol. 293, No. 28 (2018)
       
  • The Hippo pathway effector proteins YAP and TAZ have both distinct and
           overlapping functions in the cell [Signal Transduction]
    • Authors: Steven W. Plouffe; Kimberly C. Lin, Jerrell L. Moore 3rd, Frederick E. Tan, Shenghong Ma, Zhen Ye, Yunjiang Qiu, Bing Ren, Kun-Liang Guan
      Pages: 11230 - 11240
      Abstract: The Hippo pathway plays an important role in regulating tissue homeostasis, and its effectors, the transcriptional co-activators Yes-associated protein (YAP) and WW domain–containing transcription regulator 1 (WWTR1 or TAZ), are responsible for mediating the vast majority of its physiological functions. Although YAP and TAZ are thought to be largely redundant and similarly regulated by Hippo signaling, they have developmental, structural, and physiological differences that suggest they may differ in their regulation and downstream functions. To better understand the functions of YAP and TAZ in the Hippo pathway, using CRISPR/Cas9, we generated YAP KO, TAZ KO, and YAP/TAZ KO cell lines in HEK293A cells. We evaluated them in response to many environmental conditions and stimuli and used RNA-Seq to compare their transcriptional profiles. We found that YAP inactivation has a greater effect on cellular physiology (namely, cell spreading, volume, granularity, glucose uptake, proliferation, and migration) than TAZ inactivation. However, functional redundancy between YAP and TAZ was also observed. In summary, our findings confirm that the Hippo pathway effectors YAP and TAZ are master regulators for multiple cellular processes but also reveal that YAP has a stronger influence than TAZ.
      PubDate: 2018-07-13T00:10:27-07:00
      DOI: 10.1074/jbc.RA118.002715
      Issue No: Vol. 293, No. 28 (2018)
       
  • Phosphorylation-mediated structural changes within the SOAR domain of
           stromal interaction molecule 1 enable specific activation of distinct Orai
           channels. [Additions and Corrections]
    • Authors: Jill L. Thompson; Yue Lai-Zhao, Peter B. Stathopulos, Alan Grossfield, Trever J. Shuttleworth
      Pages: 11241 - 11241
      Abstract: VOLUME 293 (2018) PAGES 3145–3155Yue Zhao should be changed to Yue Lai-Zhao, as shown in the above author line.
      PubDate: 2018-07-13T00:10:27-07:00
      DOI: 10.1074/jbc.AAC118.004562
      Issue No: Vol. 293, No. 28 (2018)
       
  • The epigenetic regulator SIRT7 guards against mammalian cellular
           senescence induced by ribosomal DNA instability [Cell Biology]
    • Authors: Silvana Paredes; Maria Angulo-Ibanez, Luisa Tasselli, Scott M. Carlson, Wei Zheng, Tie-Mei Li, Katrin F. Chua
      Pages: 11242 - 11250
      Abstract: In the yeast Saccharomyces cerevisiae, genomic instability in rDNA repeat sequences is an underlying cause of cell aging and is suppressed by the chromatin-silencing factor Sir2. In humans, rDNA instability is observed in cancers and premature aging syndromes, but its underlying mechanisms and functional consequences remain unclear. Here, we uncovered a pivotal role of sirtuin 7 (SIRT7), a mammalian Sir2 homolog, in guarding against rDNA instability and show that this function of SIRT7 protects against senescence in primary human cells. We found that, mechanistically, SIRT7 is required for association of SNF2H (also called SMARCA5, SWI/SNF-related matrix-associated actin-dependent regulator of chromatin, subfamily A, member 5), a component of the nucleolar heterochromatin-silencing complex NoRC, with rDNA sequences. Defective rDNA–heterochromatin silencing in SIRT7-deficient cells unleashed rDNA instability, with excision and loss of rDNA gene copies, which in turn induced acute senescence. Mounting evidence indicates that accumulation of senescent cells significantly contributes to tissue dysfunction in aging-related pathologies. Our findings identify rDNA instability as a driver of mammalian cellular senescence and implicate SIRT7-dependent heterochromatin silencing in protecting against this process.
      PubDate: 2018-07-13T00:10:27-07:00
      DOI: 10.1074/jbc.AC118.003325
      Issue No: Vol. 293, No. 28 (2018)
       
  • A sirtuin's role in preventing senescence by protecting ribosomal DNA
           [Cell Biology]
    • Authors: Jean-Pierre Etchegaray; Raul Mostoslavsky
      Pages: 11251 - 11252
      Abstract: Ribosomes are encoded by many copies of ribosomal DNA (rDNA) packed into the nucleolus. High rates of transcription combined with highly repetitive sequences render rDNA loci particularly vulnerable to genomic instability, a proposed underlying cause of cellular senescence. The molecular mechanisms that maintain rDNA stability have remained unclear. A new paper elucidates a sirtuin-dependent mechanism that protects rDNA loci from genomic instability and prevents cellular senescence via heterochromatin silencing mediated by the chromatin remodeler SNF2H. This finding extends our understanding of chromatin dynamics within rDNA regions and offers new mechanistic insights into aging-related pathologies associated with genomic instability.
      PubDate: 2018-07-13T00:10:27-07:00
      DOI: 10.1074/jbc.H118.004040
      Issue No: Vol. 293, No. 28 (2018)
       
  • MP-4 contributes to snake venom neutralization by Mucuna pruriens seeds
           
    • Authors: Ashish Kumar; Chitra Gupta, Deepak T. Nair, Dinakar M. Salunke
      Pages: 11253 - 11253
      Abstract: VOLUME 291 (2016) PAGES 11373–11384The publisher of the Journal of Biological Chemistry is issuing an Expression of Concern to inform readers that credible concerns have been raised regarding the crystallography data shown in this paper. The Journal of Biological Chemistry will provide additional information as it becomes available.
      PubDate: 2018-07-13T00:10:27-07:00
      DOI: 10.1074/jbc.EC118.001735
      Issue No: Vol. 293, No. 28 (2018)
       
  • Looking back at the last two years: Coming home to JBC [Editorial]
    • Authors: Lila M. Gierasch
      Pages: 11254 - 11254
      Abstract: When I was approached about putting my name in the hat for the Editor-in-Chief position at JBC, a mini-tsunami of thoughts splashed through my brain. First and foremost: Why in heck would I do that' Mulling that question over led to some clarity about why I might take this position and a growing sense of anticipated pleasure at the prospect of making some small contribution to the future vigor of a society journal that I valued in the deepest part of my core. Mind you, I had not been publishing frequently in JBC in the years prior to this introspective moment. In fact, this was presented to me as a key question by the Search Committee. In answering this question, I gained insight into the challenges that journals like JBC face in the current scientific publishing climate, and what considerations come into play as prospective authors decide where to publish. PIs see a staggering array of choices before them when they prepare a submission. The junior colleagues whose careers we are fostering believe, right or wrong, that their future may be determined by their record of publishing in “high impact” journals, especially the three big single-word journals. Truth be told, most senior scientists also live under the threat, real or not, of success or failure based on “high impact” publications.But, there are other factors that weigh into the choice of journal to submit to. And, believe it or not, we do not urge anyone to submit ALL of their papers...
      PubDate: 2018-07-13T00:10:27-07:00
      DOI: 10.1074/jbc.E118.004624
      Issue No: Vol. 293, No. 28 (2018)
       
 
 
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