Journal of Biological Chemistry Impact Factor & Information

Publisher: American Society for Biochemistry and Molecular Biology, American Society for Biochemistry and Molecular Biology

Journal description

Complete content of the Journal of Biological Chemistry as of April 1995.

Current impact factor: 4.60

Impact Factor Rankings

2015 Impact Factor Available summer 2015
2013 / 2014 Impact Factor 4.6
2012 Impact Factor 4.651
2011 Impact Factor 4.773
2010 Impact Factor 5.328
2009 Impact Factor 5.328
2008 Impact Factor 5.52
2007 Impact Factor 5.581
2006 Impact Factor 5.808
2005 Impact Factor 5.854
2004 Impact Factor 6.355
2003 Impact Factor 6.482
2002 Impact Factor 6.696
2001 Impact Factor 7.258
2000 Impact Factor 7.368
1999 Impact Factor 7.666
1998 Impact Factor 7.199
1997 Impact Factor 6.963
1996 Impact Factor 7.452
1995 Impact Factor 7.385
1994 Impact Factor 7.716
1993 Impact Factor 6.793
1992 Impact Factor 6.733

Impact factor over time

Impact factor

Additional details

5-year impact 5.02
Cited half-life 10.00
Immediacy index 0.94
Eigenfactor 0.68
Article influence 1.95
Website Journal of Biological Chemistry website
Other titles Journal of biological chemistry (Online), Journal of biological chemistry, JBC online, JBC
ISSN 1083-351X
OCLC 32808313
Material type Document, Periodical, Internet resource
Document type Internet Resource, Computer File, Journal / Magazine / Newspaper

Publisher details

American Society for Biochemistry and Molecular Biology

  • Pre-print
    • Author cannot archive a pre-print version
  • Post-print
    • Author cannot archive a post-print version
  • Restrictions
    • 12 months embargo
  • Conditions
    • Authors accepted peer-reviewed manuscript may be posted on an institutional repository
    • Publisher copyright and source must be acknowledged with set phrase: "This research was originally published in Journal Name. Author(s). Title. Journal Name. Year. Vol:pp-pp. © the American Society for Biochemistry and Molecular Biology"
    • On a non-profit server
    • Publisher's version/PDF cannot be used
  • Classification
    ​ white

Publications in this journal

  • Yasunori Noguchi, Yukari Sakiyama, Hironori Kawakami, Tsutomu Katayama
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    ABSTRACT: ATP-DnaA binds to multiple DnaA boxes in the E. coli replication origin (oriC) and forms left-half and right-half subcomplexes that promote DNA unwinding and DnaB helicase loading. DnaA forms homo-oligomers in a head-to-tail manner via interactions between the bound ATP and Arg285 of the adjacent protomer. DnaA boxes R1 and R4 reside at the outer edges of the DnaA-binding region and have opposite orientations. In this study, roles for the protomers bound at R1 and R4 were elucidated using chimeric DnaA molecules that have alternative DNA binding-sequence specificity and chimeric oriC molecules bearing the alternative DnaA-binding sequence at R1 or R4. In vitro, protomers at R1 and R4 promoted initiation regardless of whether the bound nucleotide was ADP or ATP. Arg285 was shown to play an important role in the formation of subcomplexes that were active in oriC unwinding and DnaB loading. The results of in vivo analysis using the chimeric molecules were consistent with the in vitro data. Taken together, the data suggest a model in which DnaA subcomplexes form in symmetrically opposed orientations and in which the Arg285 fingers face inwards to mediate interactions with adjacent protomers. This mode is consistent with initiation regulation by ATP-DnaA and bidirectional loading of DnaB helicases. Copyright © 2015, The American Society for Biochemistry and Molecular Biology.
    Journal of Biological Chemistry 06/2015; DOI:10.1074/jbc.M115.662601
  • Merce Garcia-Belinchon, Maria Sanchez-Osuna, Laura Martinez-Escardo, Carla Granados-Colomina, Sonia Pascual-Guiral, Victoria Iglesias-Guimarais, Elisenda Casanelles, Judit Ribas, Victor J Yuste
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    ABSTRACT: Apoptosis is triggered by the activation of caspases and characterized by chromatin condensation and nuclear fragmentation (type II nuclear morphology). Necrosis is depicted by a gain in cell volume (oncosis), swelling of organelles, plasma membrane leakage and subsequent loss of intracellular contents. Although considered as different cell death entities, there is an overlap between apoptosis and necrosis. In this sense, mounting evidences suggest that both processes can be morphological expressions of a common biochemical network known as ″apoptosis-necrosis continuum″. To gain insight into the events driving the ″apoptosis-necrosis continuum″, apoptotic-proficient cells were screened facing several apoptotic inducers for the absence of type II apoptotic nuclear morphologies. Chelerythrine was selected for further studies based on its cytotoxicity and the lack of apoptotic nuclear alterations. Chelerythrine triggered an early plasma membrane leakage without condensed chromatin aggregates. Ultrastructural analysis revealed that chelerythrine-mediated cytotoxicity was compatible with a necrotic-like type of cell death. Biochemically, chelerythrine induced the activation of caspases. Moreover, the inhibition of caspases prevented chelerythrine-triggered necrotic-like cell death. Compared with staurosporine, chelerythrine induced stronger caspase activation detectable at earlier times. After employing a battery of chemicals, we found that high concentrations of thiolic antioxidants fully prevented chelerythrine-driven caspase-activation and necrotic-like cell death. Reduced amounts of thiolic antioxidants partially avoided chelerythrine-mediated cytotoxicity and allowed cells to display type II apoptotic nuclear morphology correlating with a delay in caspase-3 activation. Altogether, these data support that an early and pronounced activation of caspases can drive cells to undergo a form of necrotic-like regulated cell death. Copyright © 2015, The American Society for Biochemistry and Molecular Biology.
    Journal of Biological Chemistry 06/2015; DOI:10.1074/jbc.M115.644179
  • Ying Chen, Hai-Sheng Zhang, Guo-Hua Fong, Qiu-Lei Xi, Guo-Hao Wu, Chen-Guang Bai, Zhi-Qiang Ling, Li Fan, Yi-Ming Xu, Yan-Qing Qin, Tang-Long Yuan, Heng Sun, Jing Fang
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    ABSTRACT: Prolyl hydroxylases (PHDs) control cellular adaptation to hypoxia. PHDs are found involved in inflammatory bowel diseases (IBD); however, the exact role of PHD3, a member of PHD family, in IBD remains unknown. We show here that PHD3 plays a critical role in maintaining intestinal epithelial barrier function. We found that genetic ablation of Phd3 in intestinal epithelial cells led to spontaneous colitis in mice. Deletion of PHD3 decreases the level of tight junction protein occludin, leading to a failure of intestinal epithelial barrier function. Further studies indicate that PHD3 stabilizes occludin through preventing the interaction between the E3 ligase Itch and occludin, in a hydroxylase-independent manner. Examination of biopsy of human ulcerative colitis patients indicates that PHD3 is decreased with disease severity, indicating that PHD3 downregulation is associated with progression of this disease. We show that PHD3 protects intestinal epithelial barrier function, and reveal a hydroxylase-independent function of PHD3 in stabilizing occludin. These findings may help open avenues for developing therapeutic strategy of IBD. Copyright © 2015, The American Society for Biochemistry and Molecular Biology.
    Journal of Biological Chemistry 06/2015; DOI:10.1074/jbc.M115.653584
  • Hongwei Ma, Michael R Butler, Arjun Thapa, Josh Belcher, Fan Yang, Wolfgang Baehr, Martin Biel, Stylianos Michalakis, Xi-Qin Ding
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    ABSTRACT: Photoreceptor cyclic nucleotide-gated (CNG) channels play a pivotal role in phototransduction. Mutations in the cone CNG channel subunits CNGA3 and CNGB3 are associated with achromatopsia and cone dystrophies. We have shown endoplasmic reticulum (ER) stress-associated apoptotic cone death and increased phosphorylation of the ER Ca(2+) channel inositol 1,4,5-trisphosphate receptor 1 (IP3R1) in CNG channel-deficient mice. We also presented a remarkable elevation of cyclic guanosine monophosphate (cGMP) and an increased activity of the cGMP-dependent protein kinase (protein kinase G, PKG) in CNG channel deficiency. The current work investigated whether cGMP/PKG signaling regulates ER stress and IP3R1 phosphorylation in CNG channel-deficient cones. Treatment with PKG inhibitor and deletion of guanylate cyclase-1 (GC1), the enzyme producing cGMP in cones, were used to suppress cGMP/PKG signaling in cone-dominant Cnga3(-/-)/Nrl(-/-) mice. We found that treatment with PKG inhibitor or deletion of GC1 effectively reduced apoptotic cone death, increased expression levels of cone proteins, and decreased activation of Muller glial cells. Furthermore, we observed significantly increased phosphorylation of IP3R1 and reduced ER stress. Our findings demonstrate a role of cGMP/PKG signaling in ER stress and ER Ca(2+) channel regulation, and provide insights into the mechanism of cone degeneration in CNG channel deficiency. Copyright © 2015, The American Society for Biochemistry and Molecular Biology.
    Journal of Biological Chemistry 06/2015; DOI:10.1074/jbc.M115.641159
  • Dina B AbuSamra, Alia Al-Kilani, Samir M Hamdan, Kosuke Sakashita, Samah Z Gadhoum, Jasmeen S Merzaban
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    ABSTRACT: Selectins (E-, P- and L-) interact with glycoprotein ligands to mediate the essential tethering/rolling step in cell transport and delivery that captures migrating cells from the circulating flow. In this work, we developed a real-time immunoprecipitation assay on a surface plasmon resonance chip that captures native glycoforms of two well-known E-selectin ligands (CD44/HCELL and PSGL-1) from hematopoietic cell extracts. Here we present a comprehensive characterization of their binding to E-selectin. We show that both ligands bind recombinant monomeric E-selectin transiently with fast-on and fast-off rates while they bind dimeric E-selectin with remarkably slow on and off rates. This binding requires the sialyl Lewis x (sLe(x)) sugar moiety to be placed on both O- and N-glycans and its association, but not dissociation, is sensitive to the salt concentration. Our results suggest a mechanism through which monomeric selectins mediate initial fast-on and fast-off kinetics to help capture cells out of the circulating shear-flow; subsequently, tight binding by dimeric/oligomeric selectins is enabled to significantly slow rolling. Copyright © 2015, The American Society for Biochemistry and Molecular Biology.
    Journal of Biological Chemistry 06/2015; DOI:10.1074/jbc.M114.629451
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    ABSTRACT: Mucin-type core 1-derived O-glycans, one of the major types of O-glycans, are highly expressed in mammary gland epithelium. Abnormal O-glycans such as Tn antigen are found in over 90% of breast cancers; however, the in vivo role of these aberrant O-glycans in the etiology of breast cancer is unclear. We generated mice with mammary epithelial specific deletion of core 1-derived O-glycans. By crossing with two spontaneous mouse breast cancer models, we determined that loss of core 1-derived O-glycans delays the onset and progression of breast cancer development. Deficiency of core 1 O-glycosylation impaired the localization of Muc1, a major O-glycoprotein, on the apical surfaces of mammary epithelium. Signaling mediated by Muc1, which is critical for breast cancer development, was also defective in the absence of core 1 O-glycans. This study reveals an unexpected role of core 1-derived O-glycans in breast cancer development in mice. Copyright © 2015, The American Society for Biochemistry and Molecular Biology.
    Journal of Biological Chemistry 06/2015; DOI:10.1074/jbc.M115.654483
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    ABSTRACT: The effects of Kil peptide from bacteriophage λ on the assembly of E. coli FtsZ into one subunit thick protofilaments were studied using combined biophysical and biochemical methods. Kil peptide has been recently identified as the factor from bacteriophage λ responsible for the inhibition of bacterial cell division during lytic cycle, targeting FtsZ polymerization. Here we show that this antagonist blocks FtsZ assembly into GTPdependent protofilaments, producing a wide distribution of smaller oligomers compared with the average size of the intact protofilaments. The shortening of FtsZ protofilaments by Kil is detectable at concentrations of the peptide in the low micromolar range, the mid-point of the inhibition being close to its apparent affinity for GDP-bound FtsZ. This antagonist not only interferes with FtsZ assembly but also reverses the polymerization reaction. The negative regulation by Kil significantly reduces the GTPase activity of FtsZ protofilaments, and FtsZ polymers assembled in GMPCPP are significantly less sensitive to Kil. Our results suggest that, at high concentrations, Kil may use an inhibition mechanism involving the sequestration of FtsZ subunits, similar to that described for other inhibitors like the SOS response protein SulA or the moonlighting enzyme OpgH. This mechanism is different from those employed by the division site selection antagonists MinC and SlmA. This work provides new insight into the inhibition of FtsZ assembly by phages, considered potential tools against bacterial infection. Copyright © 2015, The American Society for Biochemistry and Molecular Biology.
    Journal of Biological Chemistry 06/2015; DOI:10.1074/jbc.M115.653329
  • Sarah E Maguire, Seth Rhoades, Wen-Feng Chen, Arjun Sengupta, Zhifeng Yue, Jason C Lim, Claire H Mitchell, Aalim M Weljie, Amita Sehgal
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    ABSTRACT: Breakdown of the major sleep promoting neurotransmitter, γ-aminobutyric acid (GABA), in the GABA shunt generates catabolites that may enter the TCA cycle, but it is unknown whether catabolic by-products of the GABA shunt actually support metabolic homeostasis. In Drosophila, the loss of the specific enzyme that degrades GABA, GABA Transaminase (GABAT), increases sleep, and we show here that it also affects metabolism such that flies lacking GABAT fail to survive on carbohydrate media. Expression of GABAT in neurons or glia rescues this phenotype, indicating a general metabolic function for this enzyme in the brain. As GABA degradation produces two catabolic products, glutamate and succinic semialdehyde (SSA), we sought to determine which was responsible for the metabolic phenotype. Through genetic and pharmacological experiments, we determined that glutamate, rather than SSA, accounts for the metabolic phenotype of gabat mutants. This is supported by biochemical measurements of catabolites in wild-type and mutant animals. Using in vitro labeling assays, we found that inhibition of GABAT affects energetic pathways. Interestingly, we also observed that gaba mutants display a general disruption in bioenergetics as measured by altered levels of TCA cycle intermediates, NAD+/NADH, and ATP levels. Finally, we report that the effects of GABAT on sleep do not depend upon glutamate, indicating that GABAT regulates metabolic and sleep homeostasis through independent mechanisms. These data indicate a role of the GABA shunt in the development of metabolic risk and suggest that neurological disorders caused by altered glutamate or GABA may be associated with metabolic disruption. Copyright © 2015, The American Society for Biochemistry and Molecular Biology.
    Journal of Biological Chemistry 06/2015; DOI:10.1074/jbc.M114.602276
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    ABSTRACT: Inverted Formin 2 (INF2) is a formin protein with unique biochemical effects on actin. In addition to the common formin ability to accelerate actin nucleation and elongation, INF2 can also sever filaments and accelerate their depolymerization. While we understand key attributes of INF2-mediated severing, we do not understand the mechanism by which INF2 accelerates depolymerization subsequent to severing. Here, we show that INF2 can create short filaments (< 60 nm) that continuously turnover actin subunits through a combination of barbed end elongation, severing, and WH2 motif-mediated depolymerization. This pseudo-steady state condition occurs whether starting from actin filaments or monomers. The rate-limiting step of the cycle is nucleotide exchange of ADP for ATP on actin monomers after release from the INF2/actin complex. Profilin addition has two effects: 1) to accelerate filament turnover six-fold by accelerating nucleotide exchange; and 2) to shift the equilibrium toward polymerization, resulting in longer filaments. In sum, our findings show that the combination of multiple interactions between INF2 and actin can work in concert to increase ATP turnover rate. Depending on the ratio of INF2:actin, this increased flux can result in rapid filament depolymerization or maintenance of short filaments. We also show that high concentrations of cytochalasin D accelerate ATP turnover by actin, but through a different mechanism from that of INF2. Copyright © 2015, The American Society for Biochemistry and Molecular Biology.
    Journal of Biological Chemistry 06/2015; DOI:10.1074/jbc.M115.670166
  • Daniel E Cooper, Pamela A Young, Eric L Klett, Rosalind A Coleman
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    ABSTRACT: Meeting the complex physiological demands of mammalian life requires strict control of the metabolism of long-chain fatty acyl-CoAs because of the multiplicity of their cellular functions. Acyl-CoAs are substrates for energy production; stored within lipid droplets as triacylglycerol, cholesterol esters, and retinol esters; esterified to form membrane phospholipids; or used to activate transcriptional and signaling pathways. Indirect evidence suggests that acyl-CoAs do not wander freely within cells, but instead, are channeled into specific pathways. In this review, we will discuss the evidence for acyl-CoA compartmentalization, highlight the key modes of acyl-CoA regulation, and diagram potential mechanisms for controlling acyl-CoA partitioning. Copyright © 2015, The American Society for Biochemistry and Molecular Biology.
    Journal of Biological Chemistry 06/2015; DOI:10.1074/jbc.R115.663260
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    ABSTRACT: MicroRNAs (miRs) are a class of small regulatory RNAs that have been implicated in diverse biological pathways, including cancer. miR-17/20a encoded by the c13orf25 locus is among the first miRs discovered to have oncogenic functions. The E2F family members have been established as the targets for these oncomiRs, which forms a negative feedback loop to control cell cycle progression. However, this pathway does not seem to be sufficient to account for elevated expression of these oncomiRs in cancer cells to promote tumorigenesis. Here we report that miR-17/20a targets a p53 activating kinase DAPK3, leading to p53-dependent transcriptional de-repression of the oncomiRs. We demonstrate that DAPK3 plays a central role in preventing miR-17/20a depletion-induced genome instability and in miR-17/20a overexpression-triggered tumor formation. This newly identified tumorigenic pathway may thus contribute to miR-17/20a amplification and tumor growth in diverse human cancers. Copyright © 2015, The American Society for Biochemistry and Molecular Biology.
    Journal of Biological Chemistry 06/2015; DOI:10.1074/jbc.M115.661504
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    ABSTRACT: The molecular characterization of symbionts is pivotal for understanding the cross-talk between symbionts and hosts. In addition to valuable knowledge obtained from symbiont genomic studies, the biochemical characterization of symbionts is important to fully understand symbiotic interactions. The bean bug (Riptortus pedestris) has been recognized as a useful experimental insect gut symbiosis model system due to its cultivable Burkholderia symbionts. This system is greatly advantageous because it allows for acquisition of a large quantity of homogeneous symbionts from the host midgut. Using these naïve gut symbionts, it was possible to directly compare in vivo symbiotic cells with in vitro cultured cells using biochemical approaches. With the goal of understanding molecular changes that occur in Burkholderia cells as they adapt to the Riptortus gut environment, we first elucidated that symbiotic Burkholderia cells are highly susceptible to purified Riptortus antimicrobial peptides. In search of the mechanisms of the increased immunosusceptibility of symbionts, we found striking differences in the cell envelope structures between cultured and symbiotic Burkholderia cells. The bacterial lipopolysaccharide O-antigen was absent from symbiotic cells examined by gel electrophoretic and mass spectrometric analyses, and their membranes were more sensitive to the detergent lysis. These changes in the cell envelope were responsible for the increased susceptibility of the Burkholderia symbionts to host innate immunity. Our results suggest that the symbiotic interactions between the Riptortus host and Burkholderia gut symbionts induce bacterial cell envelope changes to achieve successful gut symbiosis. Copyright © 2015, The American Society for Biochemistry and Molecular Biology.
    Journal of Biological Chemistry 06/2015; DOI:10.1074/jbc.M115.651158
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    ABSTRACT: The insecticidal feature of the three-domain Cry δ-endotoxins from Bacillus thuringiensis is generally attributed to their capability to form oligomeric pores, causing lysis of target larval midgut cells. However, molecular description of their oligomerization process has not been clearly defined. Here, a stable pre-pore of the 65-kDa trypsin-activated Cry4Ba mosquito-specific toxin was established through membrane-mimetic environments by forming a ~200-kDa OG (octyl-β-D-glucoside) micelle-induced trimer. The SDS-resistant trimer caused cytolysis to Sf9 insect cells-expressing Aedes-mALP (a Cry4Ba receptor) and was more effective than toxin monomer on membrane perturbation of calcein-loaded liposomes. Three-dimensional (3D) model of toxin trimer obtained by negative-stain electron microscopy (EM) in combination with single-particle reconstruction at ~5 nm resolution showed a propeller-shaped structure with three-fold symmetry. Fitting the 3D-reconstructed EM map with a 100-ns MD simulated Cry4Ba structure interacting with an OG micelle showed relative positioning of individual domains in the context of the trimeric complex with a major protrusion from the pore-forming domain. Moreover, high-speed atomic force microscopy (HS-AFM) imaging at nm-resolution and sub-second frame rate demonstrated conformational transitions from propeller-like to globular-shaped trimer upon lipid membrane interactions, implying pre-pore to pore conversion. Real-time trimeric arrangement of monomers associated with DMPC/CHAPSO-bicelle membranes was also evidently envisaged by successive HS-AFM imaging, depicting interactions among three individual subunits toward trimer formation. Altogether, our data provide the first pivotal insights into the structural requirement of membrane-induced conformational changes of Cry4Ba toxin monomers for molecular assembly of a pre-pore trimer capable of inserting into target membranes to generate a lytic pore. Copyright © 2015, The American Society for Biochemistry and Molecular Biology.
    Journal of Biological Chemistry 06/2015; DOI:10.1074/jbc.M114.627554
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    ABSTRACT: Partial degradation of the p100 subunit to generate p52 subunit is a hallmark of the alternative NF-κB pathway, which has been implicated in cancer. Here, we uncovered a role of the p97-Npl4-Ufd1 complex in mediating p100-to-p52 processing and therefore positively regulating the alternative NF-κB pathway. We observed an elevation of p97 mRNA levels in lymphoma patients, which positively correlates with NFKB2 expression, a downstream target gene of the alternative NF-κB pathway. Moreover, NFKB2 mRNA levels were aberrantly down-regulated in patients with inclusion body myopathy associated with Paget's disease of the bone and frontotemporal dementia (IBMPFD), a disease caused by mutation of p97. Inactivation of p97 or depletion of the p97-Npl4-Ufd1 complex inhibits the processing of p100 into p52, decreasing transcription of the downstream target genes. Further analyses reveal that the p97-Npl4-Ufd1 complex interacts with F-box and WD repeats protein SCFβTrCP complex to regulate the partial degradation of p100, a process involving K48- and K11-linked ubiquitination. In line with this, in LPS-induced lung damage mice model, generation of p52 is significantly decreased in p97-KD mice compared to mock mice. Finally, abrogation of p97 ATPase activity by its specific inhibitor DBeQ, efficiently decreased proliferation of lymphoma cells. Collectively, our study revealed a regulatory role of the p97-Npl4-Ufd1 complex in regulating p100 partial degradation, highlighting the potential of p97 as a drug target for cancers with aberrant activation of the alternative NF-κB pathway. Copyright © 2015, The American Society for Biochemistry and Molecular Biology.
    Journal of Biological Chemistry 06/2015; DOI:10.1074/jbc.M114.630061
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    ABSTRACT: Conjugation of plasmid pLS20 from Bacillus subtilis is limited to a time window between early and late exponential growth. Genetic evidence has suggested that pLS20-encoded protein RcoLS20 represses expression of a large conjugation operon, while Rap protein RapLS20 relieves repression. We show that RapLS20 is a true antirepressor protein that forms dimers in vivo and in vitro, and that directly binds to the repressor protein RcoLS20, in a 1:1 stoichiometry. We provide evidence that RapLS20 binds to the helix-turn-helix containing domain of RcoLS20 in vivo, likely obstructing DNA-binding of RcoLS20 as seen in competitive DNA binding experiments. The activity of RapLS20 in turn is counteracted by the addition of the cognate PhrLS20 peptide, which directly binds to the Rap protein and presumably induces a conformational change of the antirepressor. Thus, a Rap protein acts directly as an antirepressor protein during regulation of plasmid conjugation, turning on conjugation, and is counteracted by the PhrLS20 peptide, which by analogy to known Rap/Phr systems is secreted and taken back up into the cells, mediating cell density-driven regulation. Finally, we show that this switch-like process establishes a population-heterogeneity, where up to 30% of the cells induce transcription of the conjugation operon. Copyright © 2015, The American Society for Biochemistry and Molecular Biology.
    Journal of Biological Chemistry 06/2015; DOI:10.1074/jbc.M115.664110
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    ABSTRACT: The hypoxia-inducible factor (HIF) hydroxylases regulate hypoxia-sensing in animals. In humans they comprise three prolyl hydroxylases (PHDs, EGLN1-3), and factor inhibiting HIF (FIH). FIH is an asparaginyl hydroxylase catalyzing post-translational modification of HIF-α, resulting in reduction of HIF-mediated transcription. Like the PHDs, FIH is proposed to have a hypoxia-sensing role in cells, enabling responses to changes in cellular O2 availability. PHD2, the most important human PHD isoform, is proposed to be biochemically/kinetically suited as a hypoxia-sensor due to its relatively high sensitivity to changes in O2 concentration and slow reaction with O2. To ascertain whether these parameters are conserved amongst the HIF hydroxylases, we compared the reactions of FIH and PHD2 with O2. Consistent with previous reports we found lower Km (app)(O2) values for FIH than for PHD2 with all HIF-derived substrates. Under pre-steady-state conditions, the O2-initiated FIH reaction is significantly faster than that of PHD2. We then investigated the kinetics with respect to O2 of the FIH reaction with ankyrin repeat domain substrates (ARDs). FIH has lower Km (app)(O2) values for the tested ARDs than HIF-α substrates and pre-steady-state O2-initiated reactions were faster with ARDs than with HIF-α substrates. The results correlate with cellular studies showing that FIH is active at lower O2 concentrations than the PHDs, and suggest that competition between HIF-α and ARDs for FIH is likely to be biologically relevant, particularly in hypoxic conditions. The overall results are consistent with the proposal that the kinetic properties of individual oxygenases reflect their biological capacity to act as hypoxia-sensors. Copyright © 2015, The American Society for Biochemistry and Molecular Biology.
    Journal of Biological Chemistry 06/2015; DOI:10.1074/jbc.M115.653014
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    ABSTRACT: Elevated circulating free fatty acid (FFA) levels are important contributors to insulin resistance in the muscle and liver, but the underlying mechanisms require further elucidation. Here, we show that geranylgeranyl diphosphate synthase 1 (GGPPS), which is a branch-point enzyme in the mevalonic acid pathway, promotes lipid-induced muscle insulin resistance through activation of the RhoA/Rho-kinase signaling pathway. We have found that metabolic perturbation would increase GGPPS expression in the skeletal muscles of db/db mice and high-fat diets (HFD) fed mice. To address the metabolic effects of GGPPS activity in skeletal muscle, we generated mice with specific GGPPS deletions in their skeletal muscle tissue. Heterozygous knockout of GGPPS in the skeletal muscle improved systemic insulin sensitivity and glucose homeostasis in mice fed both normal chow and high-fat diets. These metabolic alterations were accompanied by activated PI3K/Akt signaling and enhanced glucose uptake in the skeletal muscle. Further investigations showed that the FFA-stimulated GGPPS expression in the skeletal muscle was able to enhance the geranylgeranylation of RhoA, which further induced the inhibitory phosphorylation of IRS-1 (Ser307) by increasing Rho-kinase activity. These results implicate a crucial role of the GGPPS/RhoA/Rho-kinase/IRS-1 pathway in skeletal muscle, in which it mediates lipid-induced systemic insulin resistance in obese mice. Therefore, skeletal muscle GGPPS may represent a potential pharmacological target for the prevention and treatment of obesity-related type 2 diabetes. Copyright © 2015, The American Society for Biochemistry and Molecular Biology.
    Journal of Biological Chemistry 06/2015; DOI:10.1074/jbc.M115.657742
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    ABSTRACT: Gene-environment interactions determine the biological outcomes through mechanisms that are poorly understood. Mouse embryonic eyelid closure is a well-defined model to study the genetic control of developmental programs. Using this model, we investigated how exposure to dioxin-like environmental pollutants modifies the genetic risk of developmental abnormalities. Our studies reveal that MAP3K1 signaling is a focal point of gene-environment crosstalk. Dioxin exposure, acting through the aryl hydrocarbon receptor (AHR), blocks eyelid closure in genetic mutants in which MAP3K1 signaling is attenuated, but does not disturb this developmental program in either wild type or mutant mice with attenuated EGFR or WNT signaling. Exposure also markedly inhibits c-Jun phosphorylation in Map3k1+/- embryonic eyelid epithelium, suggesting that dioxin-induced AHR pathways can synergize with gene mutations to inhibit MAP3K1 signaling. Our studies uncover a novel mechanism through which the dioxin-AHR axis interacts with the MAP3K1 signaling pathways during fetal development and provide strong empirical evidence that environmental pollutant exposure can increase the risk of developmental abnormalities driven by specific gene alterations. Copyright © 2015, The American Society for Biochemistry and Molecular Biology.
    Journal of Biological Chemistry 06/2015; DOI:10.1074/jbc.M115.665729
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    ABSTRACT: The T cell receptor (TCR)-CD3 complex is composed of a genetically diverse αβ TCR heterodimer associated noncovalently with the invariant CD3 dimers CD3ϵγ, CDϵδ and CD3ζζ. The TCR mediates peptide-MHC recognition, while the CD3 molecules transduce activation signals to the T cell. Whereas much is known about downstream T cell signaling pathways, the mechanism whereby TCR engagement by peptide-MHC initiates signaling is poorly understood. A key to solving this problem is defining the spatial organization of the TCR-CD3 complex and the interactions between its subunits. We have applied solution NMR methods to identify the docking site for CD3 on the β chain of a human autoimmune TCR. We demonstrate a low affinity but highly specific interaction between the extracellular domains of CD3 and the TCR constant β (Cβ) domain that requires both CD3ϵγ and CD3ϵδ subunits. The mainly hydrophilic docking site, comprising 9-11 solvent accessible Cβ residues, is relatively small (~400 A2), consistent with the weak interaction between TCR and CD3 extracellular domains, and devoid of glycosylation sites. The docking site is centered on the αA and αB helices of Cβ, which are located at the base of the TCR. This positions CD3ϵγ and CD3ϵδ between the TCR and the T cell membrane, permitting us to distinguish among several possible models of TCR-CD3 association. We further correlate structural results from NMR with mutational data on TCR-CD3 interactions from cell-based assays. Copyright © 2015, The American Society for Biochemistry and Molecular Biology.
    Journal of Biological Chemistry 06/2015; DOI:10.1074/jbc.M115.663799
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    ABSTRACT: The P-Rex (phosphatidylinositol(3,4,5)-trisphosphate (PIP3)-dependent Rac exchanger) family (P-Rex1 and P-Rex2) of Rho guanine nucleotide exchange factors (Rho GEFs) activate Rac GTPases to regulate cell migration, invasion and metastasis in several human cancers. The family is unique among Rho GEFs as their activity is regulated by the synergistic binding of PIP3 and Gβγ at the plasma membrane. However, the molecular mechanism of this family of multi-domain proteins remains unclear. We report the 1.95 Å crystal structure of the catalytic P-Rex1 DH-PH tandem domain in complex with its cognate GTPase, Rac1 (Ras-related C3 botulinum toxin substrate-1). Mutations in the P-Rex1:Rac1 interface reveal a critical role of this complex in signaling downstream of receptor tyrosine kinases and G protein-coupled receptors. Structural data indicate the PIP3/Gβγ binding sites are on the opposite surface and markedly removed from the Rac1 interface, supporting a model whereby P-Rex1 binding to PIP3 and/or Gβγ releases inhibitory C-terminal domains to expose the Rac1 binding site. Copyright © 2015, The American Society for Biochemistry and Molecular Biology.
    Journal of Biological Chemistry 06/2015; DOI:10.1074/jbc.M115.660456