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Journal of Biological Chemistry
Journal Prestige (SJR): 2.672
Citation Impact (citeScore): 4
Number of Followers: 287  
 
  Full-text available via subscription Subscription journal
ISSN (Print) 0021-9258 - ISSN (Online) 1083-351X
Published by ASBMB Homepage  [3 journals]
  • Cation channel conductance and pH gating of the innate immunity factor
           APOL1 are governed by pore-lining residues within the C-terminal domain
           [Microbiology]
    • Authors: Charles Schaub; Joseph Verdi, Penny Lee, Nada Terra, Gina Limon, Jayne Raper, Russell Thomson
      Pages: 13138 - 13149
      Abstract: The human innate immunity factor apolipoprotein L-I (APOL1) protects against infection by several protozoan parasites, including Trypanosoma brucei brucei. Endocytosis and acidification of high-density lipoprotein–associated APOL1 in trypanosome endosomes leads to eventual lysis of the parasite due to increased plasma membrane cation permeability, followed by colloid-osmotic swelling. It was previously shown that recombinant APOL1 inserts into planar lipid bilayers at acidic pH to form pH-gated nonselective cation channels that are opened upon pH neutralization. This corresponds to the pH changes encountered during endocytic recycling, suggesting APOL1 forms a cytotoxic cation channel in the parasite plasma membrane. Currently, the mechanism and domains required for channel formation have yet to be elucidated, although a predicted helix-loop-helix (H-L-H) was suggested to form pores by virtue of its similarity to bacterial pore-forming colicins. Here, we compare recombinant human and baboon APOL1 orthologs, along with interspecies chimeras and individual amino acid substitutions, to identify regions required for channel formation and pH gating in planar lipid bilayers. We found that whereas neutralization of glutamates within the H-L-H may be important for pH-dependent channel formation, there was no evidence of H-L-H involvement in either pH gating or ion selectivity. In contrast, we found two residues in the C-terminal domain, tyrosine 351 and glutamate 355, that influence pH gating properties, as well as a single residue, aspartate 348, that determines both cation selectivity and pH gating. These data point to the predicted transmembrane region closest to the APOL1 C terminus as the pore-lining segment of this novel channel-forming protein.
      PubDate: 2020-09-18T00:05:38-07:00
      DOI: 10.1074/jbc.RA120.014201
      Issue No: Vol. 295, No. 38 (2020)
       
  • H2S and reactive sulfur signaling at the host-bacterial pathogen interface
           [Gene Regulation]
    • Authors: Brenna J. C. Walsh; David P. Giedroc
      Pages: 13150 - 13168
      Abstract: Bacterial pathogens that cause invasive disease in the vertebrate host must adapt to host efforts to cripple their viability. Major host insults are reactive oxygen and reactive nitrogen species as well as cellular stress induced by antibiotics. Hydrogen sulfide (H2S) is emerging as an important player in cytoprotection against these stressors, which may well be attributed to downstream more oxidized sulfur species termed reactive sulfur species (RSS). In this review, we summarize recent work that suggests that H2S/RSS impacts bacterial survival in infected cells and animals. We discuss the mechanisms of biogenesis and clearance of RSS in the context of a bacterial H2S/RSS homeostasis model and the bacterial transcriptional regulatory proteins that act as “sensors” of cellular RSS that maintain H2S/RSS homeostasis. In addition, we cover fluorescence imaging– and MS–based approaches used to detect and quantify RSS in bacterial cells. Last, we discuss proteome persulfidation (S-sulfuration) as a potential mediator of H2S/RSS signaling in bacteria in the context of the writer-reader-eraser paradigm, and progress toward ascribing regulatory significance to this widespread post-translational modification.
      PubDate: 2020-09-18T00:05:38-07:00
      DOI: 10.1074/jbc.REV120.011304
      Issue No: Vol. 295, No. 38 (2020)
       
  • Angiotensin II type 1 receptor variants alter endosomal
           receptor-{beta}-arrestin complex stability and MAPK activation [Signal
           Transduction]
    • Authors: Yubo Cao; Sahil Kumar, Yoon Namkung, Laurence Gagnon, Aaron Cho, Stephane A. Laporte
      Pages: 13169 - 13180
      Abstract: The angiotensin II (AngII) type 1 receptor (AT1R), a member of the G protein–coupled receptor (GPCR) family, signals through G proteins and β-arrestins, which act as adaptors to regulate AT1R internalization and mitogen-activated protein kinase (MAPK) ERK1/2 activation. β-arrestin–dependent ERK1/2 regulation is the subject of important studies because its spatiotemporal control remains poorly understood for many GPCRs, including AT1R. To study the link between β-arrestin–dependent trafficking and ERK1/2 signaling, we investigated three naturally occurring AT1R variants that show distinct receptor–β-arrestin interactions: A163T, T282M, and C289W. Using bioluminescence resonance energy transfer (BRET)–based and conformational fluorescein arsenical hairpin–BRET sensors coupled with high-resolution fluorescence microscopy, we show that all AT1R variants form complexes with β-arrestin2 at the plasma membrane and efficiently internalize into endosomes upon AngII stimulation. However, mutant receptors imposed distinct conformations in β-arrestin2 and differentially impacted endosomal trafficking and MAPK signaling. Notably, T282M accumulated in endosomes, but its ability to form stable complexes following internalization was reduced, markedly impairing its ability to co-traffic with β-arrestin2. We also found that despite β-arrestin2 overexpression, T282M's and C289W's residency with β-arrestin2 in endosomes was greatly reduced, leading to decreased β-arrestin–dependent ERK1/2 activation, faster recycling of receptors to the plasma membrane, and impaired AngII-mediated proliferation. Our findings reveal that naturally occurring AT1R variants alter the patterns of receptor/β-arrestin2 trafficking and suggest conformationally dependent β-arrestin–mediated MAPK activation as well as endosomal receptor–β-arrestin complex stabilization in the mitogenic response of AT1R.
      PubDate: 2020-09-18T00:05:38-07:00
      DOI: 10.1074/jbc.RA120.014330
      Issue No: Vol. 295, No. 38 (2020)
       
  • The Ca2+ channel CatSper is not activated by cAMP/PKA signaling but
           directly affected by chemicals used to probe the action of cAMP and PKA
           [Cell Biology]
    • Authors: Tao Wang; Samuel Young, Henrike Krenz, Frank Tuttelmann, Albrecht Ropke, Claudia Krallmann, Sabine Kliesch, Xu–Hui Zeng, Christoph Brenker, Timo Strunker
      Pages: 13181 - 13193
      Abstract: The sperm-specific Ca2+ channel CatSper (cation channel of sperm) controls the influx of Ca2+ into the flagellum and, thereby, the swimming behavior of sperm. A hallmark of human CatSper is its polymodal activation by membrane voltage, intracellular pH, and oviductal hormones. Whether CatSper is also activated by signaling pathways involving an increase of cAMP and ensuing activation of PKA is, however, a matter of controversy. To shed light on this question, we used kinetic ion-sensitive fluorometry, patch-clamp recordings, and optochemistry to study transmembrane Ca2+ flux and membrane currents in human sperm from healthy donors and from patients that lack functional CatSper channels. We found that human CatSper is neither activated by intracellular cAMP directly nor indirectly by the cAMP/PKA-signaling pathway. Instead, we show that nonphysiological concentrations of cAMP and membrane-permeable cAMP analogs used to mimic the action of intracellular cAMP activate human CatSper from the outside via a hitherto-unknown extracellular binding site. Finally, we demonstrate that the effects of common PKA inhibitors on human CatSper rest predominantly, if not exclusively, on off-target drug actions on CatSper itself rather than on inhibition of PKA. We conclude that the concept of an intracellular cAMP/PKA-activation of CatSper is primarily based on unspecific effects of chemical probes used to interfere with cAMP signaling. Altogether, our findings solve several controversial issues and reveal a novel ligand-binding site controlling the activity of CatSper, which has important bearings on future studies of cAMP and Ca2+ signaling in sperm.
      PubDate: 2020-09-18T00:05:38-07:00
      DOI: 10.1074/jbc.RA120.013218
      Issue No: Vol. 295, No. 38 (2020)
       
  • Amyloid precursor protein 770 is specifically expressed and released from
           platelets [Glycobiology and Extracellular Matrices]
    • Authors: Saori Miura; Akiomi Yoshihisa, Tomofumi Misaka, Takayoshi Yamaki, Takao Kojima, Masahiro Toyokawa, Kazuei Ogawa, Hiroki Shimura, Naomasa Yamamoto, Kohji Kasahara, Yasuchika Takeishi, Shinobu Kitazume
      Pages: 13194 - 13201
      Abstract: Platelets not only play an essential role in hemostasis after vascular injury but are also involved in the development of coronary artery disease (CAD) and cerebrovascular lesions. Patients with CAD and cerebral ischemia are recommended to undergo antiplatelet therapy, but they have an increased incidence of major bleeding complications. Both assessment of the platelet activation status and response to antiplatelet therapy in each patient are highly desired. β-Amyloid precursor protein (APP) 770 is expressed in vascular endothelial cells, and its extracellular region, a soluble form of APP770 (sAPP770, also called nexin-2), is proteolytically cleaved for shedding. Abundant sAPP770 is also released from activated platelets. In this study, we used peripheral blood samples from patients with CAD and control subjects and evaluated sAPP770 as a specific biomarker for platelet activation. First, the plasma levels of sAPP770 correlated well with those of the soluble form CD40 ligand (CD40L), an established biomarker for platelet activation. Additionally, flow cytometry analysis using peripheral blood cells showed that CD40L expression is up-regulated in activated T cells, whereas APP770 expression is negligible in all blood cell types except platelets. Following stimulation with collagen or ADP, aggregating platelets immediately released sAPP770. Finally, patients with dual antiplatelet therapy showed significantly lower levels of plasma sAPP770 than those with no therapy. Taken together, our data show that plasma sAPP770 could be a promising biomarker for platelet activation.
      PubDate: 2020-09-18T00:05:38-07:00
      DOI: 10.1074/jbc.RA120.012904
      Issue No: Vol. 295, No. 38 (2020)
       
  • A new model for Trypanosoma cruzi heme homeostasis depends on modulation
           of TcHTE protein expression [Metabolism]
    • Authors: Lucas Pagura; Evelyn Tevere, Marcelo L. Merli, Julia A. Cricco
      Pages: 13202 - 13212
      Abstract: Heme is an essential cofactor for many biological processes in aerobic organisms, which can synthesize it de novo through a conserved pathway. Trypanosoma cruzi, the etiological agent of Chagas disease, as well as other trypanosomatids relevant to human health, are heme auxotrophs, meaning they must import it from their mammalian hosts or insect vectors. However, how these species import and regulate heme levels is not fully defined yet. It is known that the membrane protein TcHTE is involved in T. cruzi heme transport, although its specific role remains unclear. In the present work, we studied endogenous TcHTE in the different life cycle stages of the parasite to gain insight into its function in heme transport and homeostasis. We have confirmed that TcHTE is predominantly detected in replicative stages (epimastigote and amastigote), in which heme transport activity was previously validated. We also showed that in epimastigotes, TcHTE protein and mRNA levels decrease in response to increments in heme concentration, confirming it as a member of the heme response gene family. Finally, we demonstrated that T. cruzi epimastigotes can sense intracellular heme by an unknown mechanism and regulate heme transport to adapt to changing conditions. Based on these results, we propose a model in which T. cruzi senses intracellular heme and regulates heme transport activity by adjusting the expression of TcHTE. The elucidation and characterization of heme transport and homeostasis will contribute to a better understanding of a critical pathway for T. cruzi biology allowing the identification of novel and essential proteins.
      PubDate: 2020-09-18T00:05:38-07:00
      DOI: 10.1074/jbc.RA120.014574
      Issue No: Vol. 295, No. 38 (2020)
       
  • CHOP and c-JUN up-regulate the mutant Z {alpha}1-antitrypsin, exacerbating
           its aggregation and liver proteotoxicity [Gene Regulation]
    • Authors: Sergio Attanasio; Rosa Ferriero, Gwladys Gernoux, Rossella De Cegli, Annamaria Carissimo, Edoardo Nusco, Severo Campione, Jeffrey Teckman, Christian Mueller, Pasquale Piccolo, Nicola Brunetti-Pierri
      Pages: 13213 - 13223
      Abstract: α1-Antitrypsin (AAT) encoded by the SERPINA1 gene is an acute-phase protein synthesized in the liver and secreted into the circulation. Its primary role is to protect lung tissue by inhibiting neutrophil elastase. The Z allele of SERPINA1 encodes a mutant AAT, named ATZ, that changes the protein structure and leads to its misfolding and polymerization, which cause endoplasmic reticulum (ER) stress and liver disease through a gain-of-function toxic mechanism. Hepatic retention of ATZ results in deficiency of one of the most important circulating proteinase inhibitors and predisposes to early-onset emphysema through a loss-of-function mechanism. The pathogenetic mechanisms underlying the liver disease are not completely understood. C/EBP-homologous protein (CHOP), a transcription factor induced by ER stress, was found among the most up-regulated genes in livers of PiZ mice that express ATZ and in human livers of patients homozygous for the Z allele. Compared with controls, juvenile PiZ/Chop−/− mice showed reduced hepatic ATZ and a transcriptional response indicative of decreased ER stress by RNA-Seq analysis. Livers of PiZ/Chop−/− mice also showed reduced SERPINA1 mRNA levels. By chromatin immunoprecipitations and luciferase reporter–based transfection assays, CHOP was found to up-regulate SERPINA1 cooperating with c-JUN, which was previously shown to up-regulate SERPINA1, thus aggravating hepatic accumulation of ATZ. Increased CHOP levels were detected in diseased livers of children homozygous for the Z allele. In summary, CHOP and c-JUN up-regulate SERPINA1 transcription and play an important role in hepatic disease by increasing the burden of proteotoxic ATZ, particularly in the pediatric population.
      PubDate: 2020-09-18T00:05:38-07:00
      DOI: 10.1074/jbc.RA120.014307
      Issue No: Vol. 295, No. 38 (2020)
       
  • Complex I mutations synergize to worsen the phenotypic expression of
           Leber's hereditary optic neuropathy [Molecular Bases of Disease]
    • Authors: Yanchun Ji; Juanjuan Zhang, Yuanyuan Lu, Qiuzi Yi, Mengquan Chen, Shipeng Xie, Xiaoting Mao, Yun Xiao, Feilong Meng, Minglian Zhang, Rulai Yang, Min-Xin Guan
      Pages: 13224 - 13238
      Abstract: Leber's hereditary optic neuropathy (LHON) is a maternal inheritance of eye disease because of the mitochondrial DNA (mtDNA) mutations. We previously discovered a 3866T>C mutation within the gene for the ND1 subunit of complex I as possibly amplifying disease progression for patients bearing the disease-causing 11778G>A mutation within the gene for the ND4 subunit of complex I. However, whether and how the ND1 mutation exacerbates the ND4 mutation were unknown. In this report, we showed that four Chinese families bearing both m.3866T>C and m.11778G>A mutations exhibited higher penetrances of LHON than 6 Chinese pedigrees carrying only the m.3866T>C mutation or families harboring only the m.11778G>A mutation. The protein structure analysis revealed that the m.3866T>C (I187T) and m.11778G>A (R340H) mutations destabilized the specific interactions with other residues of ND1 and ND4, thereby altering the structure and function of complex I. Cellular data obtained using cybrids, constructed by transferring mitochondria from the Chinese families into mtDNA-less (ρ°) cells, demonstrated that the mutations perturbed the stability, assembly, and activity of complex I, leading to changes in mitochondrial ATP levels and membrane potential and increasing the production of reactive oxygen species. These mitochondrial dysfunctions promoted the apoptotic sensitivity of cells and decreased mitophagy. Cybrids bearing only the m.3866T>C mutation displayed mild mitochondrial dysfunctions, whereas those harboring both m.3866T>C and m.11778G>A mutations exhibited greater mitochondrial dysfunctions. These suggested that the m.3866T>C mutation acted in synergy with the m.11778G>A mutation, aggravating mitochondrial dysfunctions and contributing to higher penetrance of LHON in these families carrying both mtDNA mutations.
      PubDate: 2020-09-18T00:05:38-07:00
      DOI: 10.1074/jbc.RA120.014603
      Issue No: Vol. 295, No. 38 (2020)
       
  • Trapping conformational states of a flavin-dependent N-monooxygenase in
           crystallo reveals protein and flavin dynamics [Enzymology]
    • Authors: Ashley C. Campbell; Kyle M. Stiers, Julia S. Martin Del Campo, Ritcha Mehra-Chaudhary, Pablo Sobrado, John J. Tanner
      Pages: 13239 - 13249
      Abstract: The siderophore biosynthetic enzyme A (SidA) ornithine hydroxylase from Aspergillus fumigatus is a fungal disease drug target involved in the production of hydroxamate-containing siderophores, which are used by the pathogen to sequester iron. SidA is an N-monooxygenase that catalyzes the NADPH-dependent hydroxylation of l-ornithine through a multistep oxidative mechanism, utilizing a C4a-hydroperoxyflavin intermediate. Here we present four new crystal structures of SidA in various redox and ligation states, including the first structure of oxidized SidA without NADP(H) or l-ornithine bound (resting state). The resting state structure reveals a new out active site conformation characterized by large rotations of the FAD isoalloxazine around the C1–′C2′ and N10–C1′ bonds, coupled to a 10-Å movement of the Tyr-loop. Additional structures show that either flavin reduction or the binding of NADP(H) is sufficient to drive the FAD to the in conformation. The structures also reveal protein conformational changes associated with the binding of NADP(H) and l-ornithine. Some of these residues were probed using site-directed mutagenesis. Docking was used to explore the active site of the out conformation. These calculations identified two potential ligand-binding sites. Altogether, our results provide new information about conformational dynamics in flavin-dependent monooxygenases. Understanding the different active site conformations that appear during the catalytic cycle may allow fine-tuning of inhibitor discovery efforts.
      PubDate: 2020-09-18T00:05:38-07:00
      DOI: 10.1074/jbc.RA120.014750
      Issue No: Vol. 295, No. 38 (2020)
       
  • Insulin signaling requires glucose to promote lipid anabolism in
           adipocytes [Metabolism]
    • Authors: James R. Krycer; Lake-Ee Quek, Deanne Francis, Armella Zadoorian, Fiona C. Weiss, Kristen C. Cooke, Marin E. Nelson, Alexis Diaz-Vegas, Sean J. Humphrey, Richard Scalzo, Akiyoshi Hirayama, Satsuki Ikeda, Futaba Shoji, Kumi Suzuki, Kevin Huynh, Corey Giles, Bianca Varney, Shilpa R. Nagarajan, Andrew J. Hoy, Tomoyoshi Soga, Peter J. Meikle, Gregory J. Cooney, Daniel J. Fazakerley, David E. James
      Pages: 13250 - 13266
      Abstract: Adipose tissue is essential for metabolic homeostasis, balancing lipid storage and mobilization based on nutritional status. This is coordinated by insulin, which triggers kinase signaling cascades to modulate numerous metabolic proteins, leading to increased glucose uptake and anabolic processes like lipogenesis. Given recent evidence that glucose is dispensable for adipocyte respiration, we sought to test whether glucose is necessary for insulin-stimulated anabolism. Examining lipogenesis in cultured adipocytes, glucose was essential for insulin to stimulate the synthesis of fatty acids and glyceride–glycerol. Importantly, glucose was dispensable for lipogenesis in the absence of insulin, suggesting that distinct carbon sources are used with or without insulin. Metabolic tracing studies revealed that glucose was required for insulin to stimulate pathways providing carbon substrate, NADPH, and glycerol 3-phosphate for lipid synthesis and storage. Glucose also displaced leucine as a lipogenic substrate and was necessary to suppress fatty acid oxidation. Together, glucose provided substrates and metabolic control for insulin to promote lipogenesis in adipocytes. This contrasted with the suppression of lipolysis by insulin signaling, which occurred independently of glucose. Given previous observations that signal transduction acts primarily before glucose uptake in adipocytes, these data are consistent with a model whereby insulin initially utilizes protein phosphorylation to stimulate lipid anabolism, which is sustained by subsequent glucose metabolism. Consequently, lipid abundance was sensitive to glucose availability, both during adipogenesis and in Drosophila flies in vivo. Together, these data highlight the importance of glucose metabolism to support insulin action, providing a complementary regulatory mechanism to signal transduction to stimulate adipose anabolism.
      PubDate: 2020-09-18T00:05:38-07:00
      DOI: 10.1074/jbc.RA120.014907
      Issue No: Vol. 295, No. 38 (2020)
       
  • Applying gene editing to tailor precise genetic modifications in plants
           [Plant Biology]
    • Authors: Joyce Van Eck
      Pages: 13267 - 13276
      Abstract: The ability to tailor alterations in genomes, including plant genomes, in a site-specific manner has been greatly advanced through approaches that reduced the complexity and time of genome sequencing along with development of gene editing technologies. These technologies provide a valuable foundation for studies of gene function, metabolic engineering, and trait modification for crop improvement. Development of genome editing methodologies began ∼20 years ago, first with meganucleases and followed by zinc finger nucleases, transcriptional activator-like effector nucleases and, most recently, clustered regulatory interspaced short palindromic repeat (CRISPR)-associated protein (Cas) (CRISPR/Cas), which is by far the most utilized method. The premise of CRISPR/Cas centers on the cleaving of one or both DNA strands by a Cas protein, an endonuclease, followed by mending of the DNA by repair mechanisms inherent in cells. Its user-friendly construct design, greater flexibility in targeting genomic regions, and cost-effective attributes have resulted in it being widely adopted and revolutionizing precise modification of the genomes of many organisms. Indeed, the CRISPR/Cas system has been utilized for gene editing in many plant species, including important food crops, such as maize, wheat, rice, and potatoes. This review summarizes the various approaches, including the most recent designs being used to make modifications from as small as a single-base-pair change to insertion of DNA fragments. On the gene expression level, strategies are presented that make it possible to knock out or modulate through activation and repression. Also discussed are prerequisites necessary for CRISPR/Cas-mediated editing as well as the current challenges.
      PubDate: 2020-09-18T00:05:38-07:00
      DOI: 10.1074/jbc.REV120.010850
      Issue No: Vol. 295, No. 38 (2020)
       
  • Discovery of a heme-binding domain in a neuronal voltage-gated potassium
           channel [Protein Structure and Folding]
    • Authors: Mark J. Burton; Joel Cresser-Brown, Morgan Thomas, Nicola Portolano, Jaswir Basran, Samuel L. Freeman, Hanna Kwon, Andrew R. Bottrill, Manuel J. Llansola-Portoles, Andrew A. Pascal, Rebekah Jukes-Jones, Tatyana Chernova, Ralf Schmid, Noel W. Davies, Nina M. Storey, Pierre Dorlet, Peter C. E. Moody, John S. Mitcheson, Emma L. Raven
      Pages: 13277 - 13286
      Abstract: The EAG (ether-à-go-go) family of voltage-gated K+ channels are important regulators of neuronal and cardiac action potential firing (excitability) and have major roles in human diseases such as epilepsy, schizophrenia, cancer, and sudden cardiac death. A defining feature of EAG (Kv10–12) channels is a highly conserved domain on the N terminus, known as the eag domain, consisting of a Per–ARNT–Sim (PAS) domain capped by a short sequence containing an amphipathic helix (Cap domain). The PAS and Cap domains are both vital for the normal function of EAG channels. Using heme-affinity pulldown assays and proteomics of lysates from primary cortical neurons, we identified that an EAG channel, hERG3 (Kv11.3), binds to heme. In whole-cell electrophysiology experiments, we identified that heme inhibits hERG3 channel activity. In addition, we expressed the Cap and PAS domain of hERG3 in Escherichia coli and, using spectroscopy and kinetics, identified the PAS domain as the location for heme binding. The results identify heme as a regulator of hERG3 channel activity. These observations are discussed in the context of the emerging role for heme as a regulator of ion channel activity in cells.
      PubDate: 2020-09-18T00:05:38-07:00
      DOI: 10.1074/jbc.RA120.014150
      Issue No: Vol. 295, No. 38 (2020)
       
  • Klp2 and Ase1 synergize to maintain meiotic spindle stability during
           metaphase I [Cell Biology]
    • Authors: Fan Zheng; Fenfen Dong, Shuo Yu, Tianpeng Li, Yanze Jian, Lingyun Nie, Chuanhai Fu
      Pages: 13287 - 13298
      Abstract: The spindle apparatus segregates bi-oriented sister chromatids during mitosis but mono-oriented homologous chromosomes during meiosis I. It has remained unclear if similar molecular mechanisms operate to regulate spindle dynamics during mitosis and meiosis I. Here, we employed live-cell microscopy to compare the spindle dynamics of mitosis and meiosis I in fission yeast cells and demonstrated that the conserved kinesin-14 motor Klp2 plays a specific role in maintaining metaphase spindle length during meiosis I but not during mitosis. Moreover, the maintenance of metaphase spindle stability during meiosis I requires the synergism between Klp2 and the conserved microtubule cross-linker Ase1, as the absence of both proteins causes exacerbated defects in metaphase spindle stability. The synergism is not necessary for regulating mitotic spindle dynamics. Hence, our work reveals a new molecular mechanism underlying meiotic spindle dynamics and provides insights into understanding differential regulation of meiotic and mitotic events.
      PubDate: 2020-09-18T00:05:38-07:00
      DOI: 10.1074/jbc.RA120.012905
      Issue No: Vol. 295, No. 38 (2020)
       
  • Catastrophic actin filament bursting by cofilin, Aip1, and coronin [Cell
           Biology]
    • Authors: Vivian W. Tang; Ambika V. Nadkarni, William M. Brieher
      Pages: 13299 - 13313
      Abstract: Cofilin is an actin filament severing protein necessary for fast actin turnover dynamics. Coronin and Aip1 promote cofilin-mediated actin filament disassembly, but the mechanism is somewhat controversial. An early model proposed that the combination of cofilin, coronin, and Aip1 disassembled filaments in bursts. A subsequent study only reported severing. Here, we used EM to show that actin filaments convert directly into globular material. A monomer trap assay also shows that the combination of all three factors produces actin monomers faster than any two factors alone. We show that coronin accelerates the release of Pi from actin filaments and promotes highly cooperative cofilin binding to actin to create long stretches of polymer with a hypertwisted morphology. Aip1 attacks these hypertwisted regions along their sides, disintegrating them into monomers or short oligomers. The results are consistent with a catastrophic mode of disassembly, not enhanced severing alone.
      PubDate: 2020-09-18T00:05:38-07:00
      DOI: 10.1074/jbc.RA120.015018
      Issue No: Vol. 295, No. 38 (2020)
       
  • Long-term effects of the proline-rich antimicrobial peptide Oncocin112 on
           the Escherichia coli translation machinery [Cell Biology]
    • Authors: Yanyu Zhu; James C. Weisshaar, Mainak Mustafi
      Pages: 13314 - 13325
      Abstract: Proline-rich antimicrobial peptides (PrAMPs) are cationic antimicrobial peptides unusual for their ability to penetrate bacterial membranes and kill cells without causing membrane permeabilization. Structural studies show that many such PrAMPs bind deep in the peptide exit channel of the ribosome, near the peptidyl transfer center. Biochemical studies of the particular synthetic PrAMP oncocin112 (Onc112) suggest that on reaching the cytoplasm, the peptide occupies its binding site prior to the transition from initiation to the elongation phase of translation, thus blocking further initiation events. We present a superresolution fluorescence microscopy study of the long-term effects of Onc112 on ribosome, elongation factor-Tu (EF-Tu), and DNA spatial distributions and diffusive properties in intact Escherichia coli cells. The new data corroborate earlier mechanistic inferences from studies in vitro. Comparisons with the diffusive behavior induced by the ribosome-binding antibiotics chloramphenicol and kasugamycin show how the specific location of each agent's ribosomal binding site affects the long-term distribution of ribosomal species between 30S and 50S subunits versus 70S polysomes. Analysis of the single-step displacements from ribosome and EF-Tu diffusive trajectories before and after Onc112 treatment suggests that the act of codon testing of noncognate ternary complexes (TCs) at the ribosomal A-site enhances the dissociation rate of such TCs from their L7/L12 tethers. Testing and rejection of noncognate TCs on a sub-ms timescale is essential to enable incorporation of the rare cognate amino acids into the growing peptide chain at a rate of ∼20 aa/s.
      PubDate: 2020-09-18T00:05:38-07:00
      DOI: 10.1074/jbc.RA120.013587
      Issue No: Vol. 295, No. 38 (2020)
       
  • Molecular determinants of release factor 2 for ArfA-mediated ribosome
           rescue [RNA]
    • Authors: Daisuke Kurita; Tatsuhiko Abo, Hyouta Himeno
      Pages: 13326 - 13337
      Abstract: Translation termination in bacteria requires that the stop codon be recognized by release factor RF1 or RF2, leading to hydrolysis of the ester bond between the peptide and tRNA on the ribosome. As a consequence, normal termination cannot proceed if the translated mRNA lacks a stop codon. In Escherichia coli, the ribosome rescue factor ArfA releases the nascent polypeptide from the stalled ribosome with the help of RF2 in a stop codon–independent manner. Interestingly, the reaction does not proceed if RF1 is instead provided, even though the structures of RF1 and RF2 are very similar. Here, we identified the regions of RF2 required for the ArfA-dependent ribosome rescue system. Introduction of hydrophobic residues from RF2 found at the interface between RF2 and ArfA into RF1 allowed RF1 to associate with the ArfA-ribosome complex to a certain extent but failed to promote peptidyl-tRNA hydrolysis, whereas WT RF1 did not associate with the complex. We also identified the key residues required for the process after ribosome binding. Our findings provide a basis for understanding how the ArfA-ribosome complex is specifically recognized by RF2 and how RF2 undergoes a conformational change upon binding to the ArfA-ribosome complex.
      PubDate: 2020-09-18T00:05:38-07:00
      DOI: 10.1074/jbc.RA120.014664
      Issue No: Vol. 295, No. 38 (2020)
       
  • 15-Keto-PGE2 acts as a biased/partial agonist to terminate PGE2-evoked
           signaling [Computational Biology]
    • Authors: Suzu Endo; Akiko Suganami, Keijo Fukushima, Kanaho Senoo, Yumi Araki, John W. Regan, Masato Mashimo, Yutaka Tamura, Hiromichi Fujino
      Pages: 13338 - 13352
      Abstract: Prostaglandin E2 (PGE2) is well-known as an endogenous proinflammatory prostanoid synthesized from arachidonic acid by the activation of cyclooxygenase-2. E type prostanoid (EP) receptors are cognates for PGE2 that have four main subtypes: EP1 to EP4. Of these, the EP2 and EP4 prostanoid receptors have been shown to couple to Gαs-protein and can activate adenylyl cyclase to form cAMP. Studies suggest that EP4 receptors are involved in colorectal homeostasis and cancer development, but further work is needed to identify the roles of EP2 receptors in these functions. After sufficient inflammation has been evoked by PGE2, it is metabolized to 15-keto-PGE2. Thus, 15-keto-PGE2 has long been considered an inactive metabolite of PGE2. However, it may have an additional role as a biased and/or partial agonist capable of taking over the actions of PGE2 to gradually terminate reactions. Here, using cell-based experiments and in silico simulations, we show that PGE2-activated EP4 receptor–mediated signaling may evoke the primary initiating reaction of the cells, which would take over the 15-keto-PGE2–activated EP2 receptor–mediated signaling after PGE2 is metabolized to 15-keto-PGE2. The present results shed light on new aspects of 15-keto-PGE2, which may have important roles in passing on activities to EP2 receptors from PGE2-stimulated EP4 receptors as a “switched agonist.” This novel mechanism may be significant for gradually terminating PGE2-evoked inflammation and/or maintaining homeostasis of colorectal tissues/cells functions.
      PubDate: 2020-09-18T00:05:38-07:00
      DOI: 10.1074/jbc.RA120.013988
      Issue No: Vol. 295, No. 38 (2020)
       
  • EGFR forms ligand-independent oligomers that are distinct from the active
           state [Molecular Biophysics]
    • Authors: Patrick O. Byrne; Kalina Hristova, Daniel J. Leahy
      Pages: 13353 - 13362
      Abstract: The human epidermal growth factor receptor (EGFR/ERBB1) is a receptor tyrosine kinase (RTK) that forms activated oligomers in response to ligand. Much evidence indicates that EGFR/ERBB1 also forms oligomers in the absence of ligand, but the structure and physiological role of these ligand-independent oligomers remain unclear. To examine these features, we use fluorescence microscopy to measure the oligomer stability and FRET efficiency for homo- and hetero-oligomers of fluorescent protein-labeled forms of EGFR and its paralog, human epidermal growth factor receptor 2 (HER2/ERBB2) in vesicles derived from mammalian cell membranes. We observe that both receptors form ligand-independent oligomers at physiological plasma membrane concentrations. Mutations introduced in the kinase region at the active state asymmetric kinase dimer interface do not affect the stability of ligand-independent EGFR oligomers. These results indicate that ligand-independent EGFR oligomers form using interactions that are distinct from the EGFR active state.
      PubDate: 2020-09-18T00:05:38-07:00
      DOI: 10.1074/jbc.RA120.012852
      Issue No: Vol. 295, No. 38 (2020)
       
  • Anterograde trafficking of ciliary MAP kinase-like ICK/CILK1 by the
           intraflagellar transport machinery is required for intraciliary retrograde
           protein trafficking [Cell Biology]
    • Authors: Kentaro Nakamura; Tatsuro Noguchi, Mariko Takahara, Yoshihiro Omori, Takahisa Furukawa, Yohei Katoh, Kazuhisa Nakayama
      Pages: 13363 - 13376
      Abstract: ICK (also known as CILK1) is a mitogen-activated protein kinase–like kinase localized at the ciliary tip. Its deficiency is known to result in the elongation of cilia and causes ciliopathies in humans. However, little is known about how ICK is transported to the ciliary tip. We here show that the C-terminal noncatalytic region of ICK interacts with the intraflagellar transport (IFT)–B complex of the IFT machinery and participates in its transport to the ciliary tip. Furthermore, total internal reflection fluorescence microscopy demonstrated that ICK undergoes bidirectional movement within cilia, similarly to IFT particles. Analysis of ICK knockout cells demonstrated that ICK deficiency severely impairs the retrograde trafficking of IFT particles and ciliary G protein–coupled receptors. In addition, we found that in ICK knockout cells, ciliary proteins are accumulated at the bulged ciliary tip, which appeared to be torn off and released into the environment as an extracellular vesicle. The exogenous expression of various ICK constructs in ICK knockout cells indicated that the IFT-dependent transport of ICK, as well as its kinase activity and phosphorylation at the canonical TDY motif, is essential for ICK function. Thus, we unequivocally show that ICK transported to the ciliary tip is required for retrograde ciliary protein trafficking and consequently for normal ciliary function.
      PubDate: 2020-09-18T00:05:38-07:00
      DOI: 10.1074/jbc.RA120.014142
      Issue No: Vol. 295, No. 38 (2020)
       
  • Modification of lipid rafts by extracellular vesicles carrying HIV-1
           protein Nef induces redistribution of amyloid precursor protein and Tau,
           causing neuronal dysfunction [Microbiology]
    • Authors: Michael Ditiatkovski; Nigora Mukhamedova, Dragana Dragoljevic, Anh Hoang, Hann Low, Tatiana Pushkarsky, Ying Fu, Irena Carmichael, Andrew F. Hill, Andrew J. Murphy, Michael Bukrinsky, Dmitri Sviridov
      Pages: 13377 - 13392
      Abstract: HIV-associated neurocognitive disorders (HANDs) are a frequent outcome of HIV infection. Effective treatment of HIV infection has reduced the rate of progression and severity but not the overall prevalence of HANDs, suggesting ongoing pathological process even when viral replication is suppressed. In this study, we investigated how HIV-1 protein Nef secreted in extracellular vesicles (exNef) impairs neuronal functionality. ExNef were rapidly taken up by neural cells in vitro, reducing the abundance of ABC transporter A1 (ABCA1) and thus cholesterol efflux and increasing the abundance and modifying lipid rafts in neuronal plasma membranes. ExNef caused a redistribution of amyloid precursor protein (APP) and Tau to lipid rafts and increased the abundance of these proteins, as well as of Aβ42. ExNef further potentiated phosphorylation of Tau and activation of inflammatory pathways. These changes were accompanied by neuronal functional impairment. Disruption of lipid rafts with cyclodextrin reversed the phenotype. Short-term treatment of C57BL/6 mice with either purified recombinant Nef or exNef similarly resulted in reduced abundance of ABCA1 and elevated abundance of APP in brain tissue. The abundance of ABCA1 in brain tissue of HIV-infected human subjects diagnosed with HAND was lower, and the abundance of lipid rafts was higher compared with HIV-negative individuals. Levels of APP and Tau in brain tissue correlated with the abundance of Nef. Thus, modification of neuronal cholesterol trafficking and of lipid rafts by Nef may contribute to early stages of neurodegeneration and pathogenesis in HAND.
      PubDate: 2020-09-18T00:05:38-07:00
      DOI: 10.1074/jbc.RA120.014642
      Issue No: Vol. 295, No. 38 (2020)
       
  • Lysocardiolipin acyltransferase regulates NSCLC cell proliferation and
           migration by modulating mitochondrial dynamics [Lipids]
    • Authors: Long Shuang Huang; Sainath R. Kotha, Sreedevi Avasarala, Michelle VanScoyk, Robert A. Winn, Arjun Pennathur, Puttaraju S. Yashaswini, Mounica Bandela, Ravi Salgia, Yulia Y. Tyurina, Valerian E. Kagan, Xiangdong Zhu, Sekhar P. Reddy, Tara Sudhadevi, Prasanth-Kumar Punathil-Kannan, Anantha Harijith, Ramaswamy Ramchandran, Rama Kamesh Bikkavilli, Viswanathan Natarajan
      Pages: 13393 - 13406
      Abstract: Lysocardiolipin acyltransferase (LYCAT), a cardiolipin (CL)-remodeling enzyme, is crucial for maintaining normal mitochondrial function and vascular development. Despite the well-characterized role for LYCAT in the regulation of mitochondrial dynamics, its involvement in lung cancer, if any, remains incompletely understood. In this study, in silico analysis of TCGA lung cancer data sets revealed a significant increase in LYCAT expression, which was later corroborated in human lung cancer tissues and immortalized lung cancer cell lines via indirect immunofluorescence and immunoblotting, respectively. Stable knockdown of LYCAT in NSCLC cell lines not only reduced CL and increased monolyso-CL levels but also reduced in vivo tumor growth, as determined by xenograft studies in athymic nude mice. Furthermore, blocking LYCAT activity using a LYCAT mimetic peptide attenuated cell migration, suggesting a novel role for LYCAT activity in promoting NSCLC. Mechanistically, the pro-proliferative effects of LYCAT were mediated by an increase in mitochondrial fusion and a G1/S cell cycle transition, both of which are linked to increased cell proliferation. Taken together, these results demonstrate a novel role for LYCAT in promoting NSCLC and suggest that targeting LYCAT expression or activity in NSCLC may provide new avenues for the therapeutic treatment of lung cancer.
      PubDate: 2020-09-18T00:05:38-07:00
      DOI: 10.1074/jbc.RA120.012680
      Issue No: Vol. 295, No. 38 (2020)
       
  • Retraction: Inhibition of histone deacetylase activity promotes invasion
           of human cancer cells through activation of urokinase plasminogen
           activator. [Withdrawals/Retractions]
    • Authors: Sai Murali Krishna Pulukuri; Bharathi Gorantla, Jasti S. Rao
      Pages: 13407 - 13407
      Abstract: VOLUME 282 (2007) PAGES 35594–35603This article has been retracted by the publisher. None of the authors could be reached for comment. The last three lanes of the actin panel from SK-N-AS cells in Fig. 2B were reused in the first three lanes of the HDAC1 panel in Fig. 7A. In Fig. 6A, the HDAC5 and HDAC7 immunoblots are the same. A portion of the input panel from Fig. 6B was reused in the input panel for siHDAC1 in Fig. 7B.
      PubDate: 2020-09-18T00:05:38-07:00
      DOI: 10.1074/jbc.RX120.015589
      Issue No: Vol. 295, No. 38 (2020)
       
  • Correction: The RNA-binding protein HuD regulates autophagosome formation
           in pancreatic {beta} cells by promoting autophagy-related gene 5
           expression. [Additions and Corrections]
    • Authors: Chongtae Kim; Wook Kim, Heejin Lee, Eunbyul Ji, Yun-Jeong Choe, Jennifer L. Martindale, Wado Akamatsu, Hideyuki Okano, Ho-Shik Kim, Suk Woo Nam, Myriam Gorospe, Eun Kyung Lee
      Pages: 13408 - 13408
      Abstract: VOLUME 289 (2013) PAGES 112–121The autophagy flux experiments shown in Figs. 2D and 4A were performed similarly, either by serial transfection of pcDNA or pHuD followed by transfection with the GFP reporter construct or by single transfection of pcDNA or pHuD. No difference was observed with either method. As no difference was observed, the HuD and actin immunoblots from Fig. 2D were mistakenly used in Fig. 4A. The corrected Fig. 4A now shows results from a single transfection rather than a serial transfection. The quantification has also been corrected. This correction does not affect the results or conclusions of this work.jbc;295/38/13408/F4F1F4Figure 4A
      PubDate: 2020-09-18T00:05:38-07:00
      DOI: 10.1074/jbc.AAC120.015676
      Issue No: Vol. 295, No. 38 (2020)
       
  • In JBC we trust [Editorials]
    • Authors: Lila M. Gierasch
      Pages: 13409 - 13409
      Abstract: Peer Review Week (September 21–25 this year) serves as an annual reminder to thank referees for their service to the scientific community. We hope we express our appreciation and acknowledge in an ongoing way the central role of peer review in science publishing, but we will take this annual reminder to say “THANK YOU” to JBC's Editorial Board Members (EBMs) and to the other reviewers who help us maintain the quality and reliability of JBC's content.The theme of this year's Peer Review Week—Trust in Peer Review (1)—hits especially close to home for those of us involved in JBC. When I began my appointment as Editor-in-Chief, we gathered input from authors and other scientists in the field as to what they thought about JBC. What are the strengths and weaknesses' How do they think JBC fits into the scientific publishing ecosystem' What do they most appreciate about engaging with JBC' The clear message we heard time and again was that researchers knew they could count on JBC to publish solid science that stands the test of time and that JBC was a journal they could trust.We also sought input from our EBMs, who bear the largest burden in reviewing papers submitted to JBC. EBMs thus play a critical role in maintaining consistency and high standards in our review process and, as a consequence, in building the trust that our authors had described. We already knew that we trusted this group to steward the contents of the journal. However, we had not...
      PubDate: 2020-09-18T00:05:38-07:00
      DOI: 10.1074/jbc.E120.015811
      Issue No: Vol. 295, No. 38 (2020)
       
 
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