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Journal of Biological Chemistry
Journal Prestige (SJR): 2.672
Citation Impact (citeScore): 4
Number of Followers: 257  
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ISSN (Print) 0021-9258 - ISSN (Online) 1083-351X
Published by ASBMB Homepage  [3 journals]
  • A small-molecule competitive inhibitor of phosphatidic acid binding by the
           AAA+ protein NSF/Sec18 blocks the SNARE-priming stage of vacuole fusion
           [Cell Biology]
    • Authors: Robert P. Sparks; Andres S. Arango, Matthew L. Starr, Zachary L. Aboff, Logan R. Hurst, David A. Rivera-Kohr, Chi Zhang, Kevin A. Harnden, Jermaine L. Jenkins, Wayne C. Guida, Emad Tajkhorshid, Rutilio A. Fratti
      Pages: 17168 - 17185
      Abstract: The homeostasis of most organelles requires membrane fusion mediated by soluble N-ethylmaleimide–sensitive factor (NSF) attachment protein receptors (SNAREs). SNAREs undergo cycles of activation and deactivation as membranes move through the fusion cycle. At the top of the cycle, inactive cis-SNARE complexes on a single membrane are activated, or primed, by the hexameric ATPase associated with the diverse cellular activities (AAA+) protein, N-ethylmaleimide-sensitive factor (NSF/Sec18), and its co-chaperone α-SNAP/Sec17. Sec18-mediated ATP hydrolysis drives the mechanical disassembly of SNAREs into individual coils, permitting a new cycle of fusion. Previously, we found that Sec18 monomers are sequestered away from SNAREs by binding phosphatidic acid (PA). Sec18 is released from the membrane when PA is hydrolyzed to diacylglycerol by the PA phosphatase Pah1. Although PA can inhibit SNARE priming, it binds other proteins and thus cannot be used as a specific tool to further probe Sec18 activity. Here, we report the discovery of a small-molecule compound, we call IPA (inhibitor of priming activity), that binds Sec18 with high affinity and blocks SNARE activation. We observed that IPA blocks SNARE priming and competes for PA binding to Sec18. Molecular dynamics simulations revealed that IPA induces a more rigid NSF/Sec18 conformation, which potentially disables the flexibility required for Sec18 to bind to PA or to activate SNAREs. We also show that IPA more potently and specifically inhibits NSF/Sec18 activity than does N-ethylmaleimide, requiring the administration of only low micromolar concentrations of IPA, demonstrating that this compound could help to further elucidate SNARE-priming dynamics.
      PubDate: 2019-11-15T00:06:22-08:00
      DOI: 10.1074/jbc.RA119.008865
      Issue No: Vol. 294, No. 46 (2019)
  • New gadget in the membrane trafficking toolbox: A novel inhibitor of SNARE
           priming [Membrane Biology]
    • Authors: Hagai Abeliovich
      Pages: 17186 - 17187
      Abstract: NSF (N-ethylmaleimide sensitive factor) and its yeast counterpart Sec18 are highly conserved homohexameric proteins that play vital roles in eukaryotic membrane trafficking. Sec18 functions by disrupting SNARE complexes formed in cis, on the same membrane. However, the molecular mechanisms of this process are poorly understood, in large part due to the lack of selective, reversible inhibitors. A new study by Sparks et al. now reports a small molecule that appears to selectively inhibit Sec18 action in an in vitro assay. Their finding now paves the way to elucidate further details of Sec18-mediated SNARE priming.
      PubDate: 2019-11-15T00:06:22-08:00
      DOI: 10.1074/jbc.H119.011334
      Issue No: Vol. 294, No. 46 (2019)
  • The RNA-binding protein SART3 promotes miR-34a biogenesis and G1 cell
           cycle arrest in lung cancer cells [RNA]
    • Authors: Emily J. Sherman; Dylan C. Mitchell, Amanda L. Garner
      Pages: 17188 - 17196
      Abstract: MicroRNAs (miRNAs or miRs) are small, noncoding RNAs that are implicated in the regulation of most biological processes. Global miRNA biogenesis is altered in many cancers, and RNA-binding proteins play a role in miRNA biogenesis, presenting a promising avenue for targeting miRNA dysregulation in diseases. miR-34a exhibits tumor-suppressive activities by targeting cell cycle regulators CDK4/6 and anti-apoptotic factor BCL-2, among other regulatory pathways such as Wnt, TGF-β, and Notch signaling. Many cancers exhibit down-regulation or loss of miR-34a, and synthetic miR-34a supplementation has been shown to inhibit tumor growth in vivo. However, the post-transcriptional mechanisms that cause miR-34a loss in cancer are not entirely understood. Here, using a proteomics-mediated approach in non-small-cell lung cancer (NSCLC) cells, we identified squamous cell carcinoma antigen recognized by T-cells 3 (SART3) as a putative pre-miR-34a–binding protein. SART3 is a spliceosome recycling factor and nuclear RNA-binding protein with no previously reported role in miRNA regulation. We found that SART3 binds pre-miR-34a with higher specificity than pre-let-7d (used as a negative control) and elucidated a new functional role for SART3 in NSCLC cells. SART3 overexpression increased miR-34a levels, down-regulated the miR-34a target genes CDK4/6, and caused a cell cycle arrest in the G1 phase. In vitro binding experiments revealed that the RNA-recognition motifs within the SART3 sequence are responsible for selective pre-miR-34a binding. Our results provide evidence for a significant role of SART3 in miR-34a biogenesis and cell cycle progression in NSCLC cells.
      PubDate: 2019-11-15T00:06:22-08:00
      DOI: 10.1074/jbc.AC119.010419
      Issue No: Vol. 294, No. 46 (2019)
  • Molecular analysis of an enigmatic Streptococcus pneumoniae virulence
           factor: The raffinose-family oligosaccharide utilization system [Protein
           Structure and Folding]
    • Authors: Joanne K. Hobbs; Edward P. W. Meier, Benjamin Pluvinage, Mackenzie A. Mey, Alisdair B. Boraston
      Pages: 17197 - 17208
      Abstract: Streptococcus pneumoniae is an opportunistic respiratory pathogen that can spread to other body sites, including the ears, brain, and blood. The ability of this bacterium to break down, import, and metabolize a wide range of glycans is key to its virulence. Intriguingly, S. pneumoniae can utilize several plant oligosaccharides for growth in vitro, including raffinose-family oligosaccharides (RFOs, which are α-(1→6)-galactosyl extensions of sucrose). An RFO utilization locus has been identified in the pneumococcal genome; however, none of the proteins encoded by this locus have been biochemically characterized. The enigmatic ability of S. pneumoniae to utilize RFOs has recently received attention because mutations in two of the RFO locus genes have been linked to the tissue tropism of clinical pneumococcal isolates. Here, we use functional studies combined with X-ray crystallography to show that although the pneumococcal RFO locus encodes for all the machinery required for uptake and degradation of RFOs, the individual pathway components are biochemically inefficient. We also demonstrate that the initiating enzyme in this pathway, the α-galactosidase Aga (a family 36 glycoside hydrolase), can cleave α-(1→3)-linked galactose units from a linear blood group antigen. We propose that the pneumococcal RFO pathway is an evolutionary relic that is not utilized in this streptococcal species and, as such, is under no selection pressure to maintain binding affinity and/or catalytic efficiency. We speculate that the apparent contribution of RFO utilization to pneumococcal tissue tropism may, in fact, be due to the essential role the ATPase RafK plays in the transport of other carbohydrates.
      PubDate: 2019-11-15T00:06:22-08:00
      DOI: 10.1074/jbc.RA119.010280
      Issue No: Vol. 294, No. 46 (2019)
  • The E3 ubiquitin ligase Pib1 regulates effective gluconeogenic shutdown
           upon glucose availability [Protein Synthesis and Degradation]
    • Authors: Vineeth Vengayil; Zeenat Rashida, Sunil Laxman
      Pages: 17209 - 17223
      Abstract: Cells use multiple mechanisms to regulate their metabolic states in response to changes in their nutrient environment. One example is the response of cells to glucose. In Saccharomyces cerevisiae growing in glucose-depleted medium, the re-availability of glucose leads to the down-regulation of gluconeogenesis and the activation of glycolysis, leading to “glucose repression.” However, our knowledge of the mechanisms mediating the glucose-dependent down-regulation of the gluconeogenic transcription factors is limited. Using the major gluconeogenic transcription factor Rds2 as a candidate, we identify here a novel role for the E3 ubiquitin ligase Pib1 in regulating the stability and degradation of Rds2. Glucose addition to cells growing under glucose limitation results in a rapid ubiquitination of Rds2, followed by its proteasomal degradation. Through in vivo and in vitro experiments, we establish Pib1 as the ubiquitin E3 ligase that regulates Rds2 ubiquitination and stability. Notably, this Pib1-mediated Rds2 ubiquitination, followed by proteasomal degradation, is specific to the presence of glucose. This Pib1-mediated ubiquitination of Rds2 depends on the phosphorylation state of Rds2, suggesting a cross-talk between ubiquitination and phosphorylation to achieve a metabolic state change. Using stable isotope-based metabolic flux experiments, we find that the loss of Pib1 results in an imbalanced gluconeogenic state, regardless of glucose availability. Pib1 is required for complete glucose repression and enables cells to optimally grow in competitive environments when glucose again becomes available. Our results reveal the existence of a Pib1-mediated regulatory program that mediates glucose repression when glucose availability is restored.
      PubDate: 2019-11-15T00:06:22-08:00
      DOI: 10.1074/jbc.RA119.009822
      Issue No: Vol. 294, No. 46 (2019)
  • Carbon source regulates polysaccharide capsule biosynthesis in
           Streptococcus pneumoniae [Glycobiology and Extracellular Matrices]
    • Authors: Lukas J. Troxler; Joel P. Werren, Thierry O. Schaffner, Nadezda Mostacci, Peter Vermathen, Martina Vermathen, Daniel Wuthrich, Cedric Simillion, Silvio D. Brugger, Remy Bruggmann, Lucy J. Hathaway, Julien Furrer, Markus Hilty
      Pages: 17224 - 17238
      Abstract: The exopolysaccharide capsule of Streptococcus pneumoniae is an important virulence factor, but the mechanisms that regulate capsule thickness are not fully understood. Here, we investigated the effects of various exogenously supplied carbohydrates on capsule production and gene expression in several pneumococcal serotypes. Microscopy analyses indicated a near absence of the capsular polysaccharide (CPS) when S. pneumoniae was grown on fructose. Moreover, serotype 7F pneumococci produced much less CPS than strains of other serotypes (6B, 6C, 9V, 15, and 23F) when grown on glucose or sucrose. RNA-sequencing revealed carbon source-dependent regulation of distinct genes of WT strains and capsule-switch mutants of serotypes 6B and 7F, but could not explain the mechanism of capsule thickness regulation. In contrast, 31P NMR of whole-cell extract from capsule-knockout strains (Δcps) clearly revealed the accumulation or absence of capsule precursor metabolites when cells were grown on glucose or fructose, respectively. This finding suggests that fructose uptake mainly results in intracellular fructose 1-phosphate, which is not converted to CPS precursors. In addition, serotype 7F strains accumulated more precursors than did 6B strains, indicating less efficient conversion of precursor metabolites into the CPS in 7F, in line with its thinner capsule. Finally, isotopologue sucrose labeling and NMR analyses revealed that the uptake of the labeled fructose subunit into the capsule is
      PubDate: 2019-11-15T00:06:22-08:00
      DOI: 10.1074/jbc.RA119.010764
      Issue No: Vol. 294, No. 46 (2019)
  • Differential regulation of AMP-activated protein kinase in healthy and
           cancer cells explains why V-ATPase inhibition selectively kills cancer
           cells [Metabolism]
    • Authors: Karin Bartel; Rolf Muller, Karin von Schwarzenberg
      Pages: 17239 - 17248
      Abstract: The cellular energy sensor AMP-activated protein kinase (AMPK) is a metabolic hub regulating various pathways involved in tumor metabolism. Here we report that vacuolar H+-ATPase (V-ATPase) inhibition differentially affects regulation of AMPK in tumor and nontumor cells and that this differential regulation contributes to the selectivity of V-ATPase inhibitors for tumor cells. In nonmalignant cells, the V-ATPase inhibitor archazolid increased phosphorylation and lysosomal localization of AMPK. We noted that AMPK localization has a prosurvival role, as AMPK silencing decreased cellular growth rates. In contrast, in cancer cells, we found that AMPK is constitutively active and that archazolid does not affect its phosphorylation and localization. Moreover, V-ATPase–independent AMPK induction in tumor cells protected them from archazolid-induced cytotoxicity, further underlining the role of AMPK as a prosurvival mediator. These observations indicate that AMPK regulation is uncoupled from V-ATPase activity in cancer cells and that this makes them more susceptible to cell death induction by V-ATPase inhibitors. In both tumor and healthy cells, V-ATPase inhibition induced a distinct metabolic regulatory cascade downstream of AMPK, affecting ATP and NADPH levels, glucose uptake, and reactive oxygen species production. We could attribute the prosurvival effects to AMPK's ability to maintain redox homeostasis by inhibiting reactive oxygen species production and maintaining NADPH levels. In summary, the results of our work indicate that V-ATPase inhibition has differential effects on AMPK-mediated metabolic regulation in cancer and healthy cells and explain the tumor-specific cytotoxicity of V-ATPase inhibition.
      PubDate: 2019-11-15T00:06:22-08:00
      DOI: 10.1074/jbc.RA119.010243
      Issue No: Vol. 294, No. 46 (2019)
  • The pseudosubstrate inhibitor Acm1 inhibits the anaphase-promoting
           complex/cyclosome by combining high-affinity activator binding with
           disruption of Doc1/Apc10 function [Cell Biology]
    • Authors: Liang Qin; Arda Mizrak, Dimitrius Santiago P. S. F. Guimaraes, Hana M. Tambrin, David O. Morgan, Mark C. Hall
      Pages: 17249 - 17261
      Abstract: The anaphase-promoting complex/cyclosome (APC/C) is a large, multisubunit ubiquitin ligase involved in regulation of cell division. APC/C substrate specificity arises from binding of short degron motifs in its substrates to transient activator subunits, Cdc20 and Cdh1. The destruction box (D-box) is the most common APC/C degron and plays a crucial role in substrate degradation by linking the activator to the Doc1/Apc10 subunit of core APC/C to stabilize the active holoenzyme and promote processive ubiquitylation. Degrons are also employed as pseudosubstrate motifs by APC/C inhibitors, and pseudosubstrates must bind their cognate activators tightly to outcompete substrate binding while blocking their own ubiquitylation. Here we examined how APC/C activity is suppressed by the small pseudosubstrate inhibitor Acm1 from budding yeast (Saccharomyces cerevisiae). Mutation of a conserved D-box converted Acm1 into an efficient ABBA (cyclin A, BubR1, Bub1, Acm1) motif–dependent APC/CCdh1 substrate in vivo, suggesting that this D-box somehow inhibits APC/C. We then identified a short conserved sequence at the C terminus of the Acm1 D-box that was necessary and sufficient for APC/C inhibition. In several APC/C substrates, the corresponding D-box region proved to be important for their degradation despite poor sequence conservation, redefining the D-box as a 12-amino acid motif. Biochemical analysis suggested that the Acm1 D-box extension inhibits reaction processivity by perturbing the normal interaction with Doc1/Apc10. Our results reveal a simple, elegant mode of pseudosubstrate inhibition that combines high-affinity activator binding with specific disruption of Doc1/Apc10 function in processive ubiquitylation.
      PubDate: 2019-11-15T00:06:22-08:00
      DOI: 10.1074/jbc.RA119.009468
      Issue No: Vol. 294, No. 46 (2019)
  • The phosphorylation status of Ser-637 in dynamin-related protein 1 (Drp1)
           does not determine Drp1 recruitment to mitochondria [Membrane Biology]
    • Authors: Rong Yu; Tong Liu, Chenfei Ning, Fei Tan, Shao–Bo Jin, Urban Lendahl, Jian Zhao, Monica Nister
      Pages: 17262 - 17277
      Abstract: Recruitment of the GTPase dynamin-related protein 1 (Drp1) to mitochondria is a central step required for mitochondrial fission. Reversible Drp1 phosphorylation has been implicated in the regulation of this process, but whether Drp1 phosphorylation at Ser-637 determines its subcellular localization and fission activity remains to be fully elucidated. Here, using HEK 293T cells and immunofluorescence, immunoblotting, RNAi, subcellular fractionation, co-immunoprecipitation assays, and CRISPR/Cas9 genome editing, we show that Drp1 phosphorylated at Ser-637 (Drp1pS637) resides both in the cytosol and on mitochondria. We found that the receptors mitochondrial fission factor (Mff) and mitochondrial elongation factor 1/2 (MIEF1/2) interact with and recruit Drp1pS637 to mitochondria and that elevated Mff or MIEF levels promote Drp1pS637 accumulation on mitochondria. We also noted that protein kinase A (PKA), which mediates phosphorylation of Drp1 on Ser-637, is partially present on mitochondria and interacts with both MIEFs and Mff. PKA knockdown did not affect the Drp1-Mff interaction, but slightly enhanced the interaction between Drp1 and MIEFs. In Drp1-deficient HEK 293T cells, both phosphomimetic Drp1-S637D and phospho-deficient Drp1-S637A variants, like wild-type Drp1, located to the cytosol and to mitochondria and rescued a Drp1 deficiency-induced mitochondrial hyperfusion phenotype. However, Drp1-S637D was less efficient than Drp1-WT and Drp1-S637A in inducing mitochondrial fission. In conclusion, the Ser-637 phosphorylation status in Drp1 is not a determinant that controls Drp1 recruitment to mitochondria.
      PubDate: 2019-11-15T00:06:22-08:00
      DOI: 10.1074/jbc.RA119.008202
      Issue No: Vol. 294, No. 46 (2019)
  • The ACT domain in chloroplast precursor-phosphorylating STY kinases binds
           metabolites and allosterically regulates kinase activity [Cell Biology]
    • Authors: Ahmed Eisa; Bettina Bolter, Serena Schwenkert
      Pages: 17278 - 17288
      Abstract: Protein import of nucleus-encoded proteins into plant chloroplasts is a highly regulated process, requiring fine-tuning mechanisms especially during chloroplast differentiation. One way of altering import efficiency is phosphorylation of chloroplast transit peptides in the cytosol. We recently investigated the role of three serine/threonine/tyrosine (STY) kinases, STY8, STY17, and STY46, in precursor phosphorylation. These three kinases have a high degree of similarity and harbor a conserved aspartate kinase–chorismate mutase–tyrA (prephenate dehydrogenase) (ACT) domain upstream of the kinase domain. The ACT domain is a widely distributed structural motif known to be important for allosteric regulation of many enzymes. In this work, using biochemical and biophysical techniques in vitro and in planta, including kinase assays, microscale thermophoresis, size exclusion chromatography, as well as site-directed mutagenesis approaches, we show that the ACT domain regulates autophosphorylation and substrate phosphorylation of the STY kinases. We found that isoleucine and S-adenosylmethionine bind to the ACT domain, negatively influencing its autophosphorylation ability. Moreover, we investigated the role of the ACT domain in planta and confirmed its involvement in chloroplast differentiation in vivo. Our results provide detailed insights into the regulation of enzyme activity by ACT domains and establish that it has a role in binding amino acid ligands during chloroplast biogenesis.
      PubDate: 2019-11-15T00:06:22-08:00
      DOI: 10.1074/jbc.RA119.010298
      Issue No: Vol. 294, No. 46 (2019)
  • Ostreolysin A and anthrolysin O use different mechanisms to control
           movement of cholesterol from the plasma membrane to the endoplasmic
           reticulum [Methods and Resources]
    • Authors: Kristen A. Johnson; Shreya Endapally, Danya C. Vazquez, Rodney E. Infante, Arun Radhakrishnan
      Pages: 17289 - 17300
      Abstract: Recent studies using two cholesterol-binding bacterial toxin proteins, perfringolysin O (PFO) and domain 4 of anthrolysin O (ALOD4), have shown that cholesterol in the plasma membranes (PMs) of animal cells resides in three distinct pools. The first pool comprises mobile cholesterol, accessible to both PFO and ALOD4, that is rapidly transported to the endoplasmic reticulum (ER) to signal cholesterol excess and maintain cholesterol homeostasis. The second is a sphingomyelin (SM)-sequestered pool inaccessible to PFO and ALOD4 but that becomes accessible by treatment with SM-degrading sphingomyelinase (SMase). The third is an essential pool also inaccessible to PFO and ALOD4 that cannot be liberated by SMase treatment. The accessible cholesterol pool can be trapped on PMs of live cells by nonlytic ALOD4, blocking its transport to the ER. However, studies of the two other pools have been hampered by a lack of available tools. Here, we used ostreolysin A (OlyA), which specifically binds SM/cholesterol complexes in membranes, to study the SM-sequestered cholesterol pool. Binding of nonlytic OlyA to SM/cholesterol complexes in PMs of live cells depleted the accessible PM cholesterol pool detectable by ALOD4. Consequently, transport of accessible cholesterol from PM to ER ceased, thereby activating SREBP transcription factors and increasing cholesterol synthesis. Thus, OlyA and ALOD4 both control movement of PM cholesterol, but through different lipid-binding mechanisms. We also found that PM-bound OlyA was rapidly internalized into cells, whereas PM-bound ALOD4 remained on the cell surface. Our findings establish OlyA and ALOD4 as complementary tools to investigate cellular cholesterol transport.
      PubDate: 2019-11-15T00:06:22-08:00
      DOI: 10.1074/jbc.RA119.010393
      Issue No: Vol. 294, No. 46 (2019)
  • Regulation of human trophoblast syncytialization by histone demethylase
           LSD1 [Developmental Biology]
    • Authors: Jessica Milano-Foster; Soma Ray, Pratik Home, Avishek Ganguly, Bhaswati Bhattacharya, Shilpika Bajpai, Aratrika Pal, Clifford W. Mason, Soumen Paul
      Pages: 17301 - 17313
      Abstract: A successful pregnancy is critically dependent upon proper placental development and function. During human placentation, villous cytotrophoblast (CTB) progenitors differentiate to form syncytiotrophoblasts (SynTBs), which provide the exchange surface between the mother and fetus and secrete hormones to ensure proper progression of pregnancy. However, epigenetic mechanisms that regulate SynTB differentiation from CTB progenitors are incompletely understood. Here, we show that lysine-specific demethylase 1 (LSD1; also known as KDM1A), a histone demethylase, is essential to this process. LSD1 is expressed both in CTB progenitors and differentiated SynTBs in first-trimester placental villi; accordingly, expression in SynTBs is maintained throughout gestation. Impairment of LSD1 function in trophoblast progenitors inhibits induction of endogenous retrovirally encoded genes SYNCYTIN1/endogenous retrovirus group W member 1, envelope (ERVW1) and SYNCYTIN2/endogenous retrovirus group FRD member 1, envelope (ERVFRD1), encoding fusogenic proteins critical to human trophoblast syncytialization. Loss of LSD1 also impairs induction of chorionic gonadotropin α (CGA) and chorionic gonadotropin β (CGB) genes, which encode α and β subunits of human chorionic gonadotrophin (hCG), a hormone essential to modulate maternal physiology during pregnancy. Mechanistic analyses at the endogenous ERVW1, CGA, and CGB loci revealed a regulatory axis in which LSD1 induces demethylation of repressive histone H3 lysine 9 dimethylation (H3K9Me2) and interacts with transcription factor GATA2 to promote RNA polymerase II (RNA-POL-II) recruitment and activate gene transcription. Our study reveals a novel LSD1–GATA2 axis, which regulates human trophoblast syncytialization.
      PubDate: 2019-11-15T00:06:23-08:00
      DOI: 10.1074/jbc.RA119.010518
      Issue No: Vol. 294, No. 46 (2019)
  • Dilated cardiomyopathy mutation in the converter domain of human cardiac
           myosin alters motor activity and response to omecamtiv mecarbil [Molecular
    • Authors: Wanjian Tang; William C. Unrath, Rohini Desetty, Christopher M. Yengo
      Pages: 17314 - 17325
      Abstract: We investigated a dilated cardiomyopathy (DCM) mutation (F764L) in human β-cardiac myosin by determining its motor properties in the presence and absence of the heart failure drug omecamtive mecarbil (OM). The mutation is located in the converter domain, a key region of communication between the catalytic motor and lever arm in myosins, and is nearby but not directly in the OM-binding site. We expressed and purified human β-cardiac myosin subfragment 1 (M2β-S1) containing the F764L mutation, and compared it to WT with in vitro motility as well as steady-state and transient kinetics measurements. In the absence of OM we demonstrate that the F764L mutation does not significantly change maximum actin-activated ATPase activity but slows actin sliding velocity (15%) and the actomyosin ADP release rate constant (25%). The transient kinetic analysis without OM demonstrates that F764L has a similar duty ratio as WT in unloaded conditions. OM is known to enhance force generation in cardiac muscle while it inhibits the myosin power stroke and enhances actin-attachment duration. We found that OM has a reduced impact on F764L ATPase and sliding velocity compared with WT. Specifically, the EC50 for OM induced inhibition of in vitro motility was 3-fold weaker in F764L. Also, OM reduces maximum actin-activated ATPase 2-fold in F764L, compared with 4-fold with WT. Overall, our results suggest that F764L attenuates the impact of OM on actin-attachment duration and/or the power stroke. Our work highlights the importance of mutation-specific considerations when pursuing small molecule therapies for cardiomyopathies.
      PubDate: 2019-11-15T00:06:23-08:00
      DOI: 10.1074/jbc.RA119.010217
      Issue No: Vol. 294, No. 46 (2019)
  • Glucoselysine is derived from fructose and accumulates in the eye lens of
           diabetic rats [Glycobiology and Extracellular Matrices]
    • Authors: Rei-ichi Ohno; Kenta Ichimaru, Seitaro Tanaka, Hikari Sugawa, Nana Katsuta, Shiori Sakake, Yu-ki Tominaga, Ikuho Ban, Jun-ichi Shirakawa, Yoshiki Yamaguchi, Emi Ito, Naoyuki Taniguchi, Ryoji Nagai
      Pages: 17326 - 17338
      Abstract: Prolonged hyperglycemia generates advanced glycation end-products (AGEs), which are believed to be involved in the pathogenesis of diabetic complications. In the present study, we developed a polyclonal antibody against fructose-modified proteins (Fru-P antibody) and identified its epitope as glucoselysine (GL) by NMR and LC-electrospray ionization (ESI)- quadrupole TOF (QTOF) analyses and evaluated its potential role in diabetes sequelae. Although the molecular weight of GL was identical to that of fructoselysine (FL), GL was distinguishable from FL because GL was resistant to acid hydrolysis, which converted all of the FLs to furosine. We also detected GL in vitro when reduced BSA was incubated with fructose for 1 day. However, when we incubated reduced BSA with glucose, galactose, or mannose for 14 days, we did not detect GL, suggesting that GL is dominantly generated from fructose. LC-ESI-MS/MS experiments with synthesized [13C6]GL indicated that the GL levels in the rat eye lens time-dependently increase after streptozotocin-induced diabetes. We observed a 31.3-fold increase in GL 8 weeks after the induction compared with nondiabetic rats, and Nϵ-(carboxymethyl)lysine and furosine increased by 1.7- and 21.5-fold, respectively, under the same condition. In contrast, sorbitol in the lens levelled off at 2 weeks after diabetes induction. We conclude that GL may be a useful biological marker to monitor and elucidate the mechanism of protein degeneration during progression of diabetes.
      PubDate: 2019-11-15T00:06:23-08:00
      DOI: 10.1074/jbc.RA119.010744
      Issue No: Vol. 294, No. 46 (2019)
  • A carbohydrate-binding family 48 module enables feruloyl esterase action
           on polymeric arabinoxylan [Protein Structure and Folding]
    • Authors: Jesper Holck; Folmer Fredslund, Marie S. Moller, Jesper Brask, Kristian B. R. M. Krogh, Lene Lange, Ditte H. Welner, Birte Svensson, Anne S. Meyer, Casper Wilkens
      Pages: 17339 - 17353
      Abstract: Feruloyl esterases (EC, belonging to carbohydrate esterase family 1 (CE1), hydrolyze ester bonds between ferulic acid (FA) and arabinose moieties in arabinoxylans. Recently, some CE1 enzymes identified in metagenomics studies have been predicted to contain a family 48 carbohydrate-binding module (CBM48), a CBM family associated with starch binding. Two of these CE1s, wastewater treatment sludge (wts) Fae1A and wtsFae1B isolated from wastewater treatment surplus sludge, have a cognate CBM48 domain and are feruloyl esterases, and wtsFae1A binds arabinoxylan. Here, we show that wtsFae1B also binds to arabinoxylan and that neither binds starch. Surface plasmon resonance analysis revealed that wtsFae1B's Kd for xylohexaose is 14.8 μm and that it does not bind to starch mimics, β-cyclodextrin, or maltohexaose. Interestingly, in the absence of CBM48 domains, the CE1 regions from wtsFae1A and wtsFae1B did not bind arabinoxylan and were also unable to catalyze FA release from arabinoxylan. Pretreatment with a β-d-1,4-xylanase did enable CE1 domain-mediated FA release from arabinoxylan in the absence of CBM48, indicating that CBM48 is essential for the CE1 activity on the polysaccharide. Crystal structures of wtsFae1A (at 1.63 Å resolution) and wtsFae1B (1.98 Å) revealed that both are folded proteins comprising structurally-conserved hydrogen bonds that lock the CBM48 position relative to that of the CE1 domain. wtsFae1A docking indicated that both enzymes accommodate the arabinoxylan backbone in a cleft at the CE1–CBM48 domain interface. Binding at this cleft appears to enable CE1 activities on polymeric arabinoxylan, illustrating an unexpected and crucial role of CBM48 domains for accommodating arabinoxylan.
      PubDate: 2019-11-15T00:06:23-08:00
      DOI: 10.1074/jbc.RA119.009523
      Issue No: Vol. 294, No. 46 (2019)
  • Interaction of the N terminus of ADP-ribosylation factor with the PH
           domain of the GTPase-activating protein ASAP1 requires
           phosphatidylinositol 4,5-bisphosphate [Signal Transduction]
    • Authors: Neeladri Sekhar Roy; Xiaoying Jian, Olivier Soubias, Peng Zhai, Jessica R. Hall, Jessica N. Dagher, Nathan P. Coussens, Lisa M. Jenkins, Ruibai Luo, Itoro O. Akpan, Matthew D. Hall, R. Andrew Byrd, Marielle E. Yohe, Paul A. Randazzo
      Pages: 17354 - 17370
      Abstract: Arf GAP with Src homology 3 domain, ankyrin repeat, and pleckstrin homology (PH) domain 1 (ASAP1) is a multidomain GTPase-activating protein (GAP) for ADP-ribosylation factor (ARF)-type GTPases. ASAP1 affects integrin adhesions, the actin cytoskeleton, and invasion and metastasis of cancer cells. ASAP1's cellular function depends on its highly-regulated and robust ARF GAP activity, requiring both the PH and the ARF GAP domains of ASAP1, and is modulated by phosphatidylinositol 4,5-bisphosphate (PIP2). The mechanistic basis of PIP2-stimulated GAP activity is incompletely understood. Here, we investigated whether PIP2 controls binding of the N-terminal extension of ARF1 to ASAP1's PH domain and thereby regulates its GAP activity. Using [Δ17]ARF1, lacking the N terminus, we found that PIP2 has little effect on ASAP1's activity. A soluble PIP2 analog, dioctanoyl-PIP2 (diC8PIP2), stimulated GAP activity on an N terminus–containing variant, [L8K]ARF1, but only marginally affected activity on [Δ17]ARF1. A peptide comprising residues 2–17 of ARF1 ([2–17]ARF1) inhibited GAP activity, and PIP2-dependently bound to a protein containing the PH domain and a 17-amino acid-long interdomain linker immediately N-terminal to the first β-strand of the PH domain. Point mutations in either the linker or the C-terminal α-helix of the PH domain decreased [2–17]ARF1 binding and GAP activity. Mutations that reduced ARF1 N-terminal binding to the PH domain also reduced the effect of ASAP1 on cellular actin remodeling. Mutations in the ARF N terminus that reduced binding also reduced GAP activity. We conclude that PIP2 regulates binding of ASAP1's PH domain to the ARF1 N terminus, which may partially regulate GAP activity.
      PubDate: 2019-11-15T00:06:23-08:00
      DOI: 10.1074/jbc.RA119.009269
      Issue No: Vol. 294, No. 46 (2019)
  • Mutations at hypothetical binding site 2 in insulin and insulin-like
           growth factors 1 and 2 result in receptor- and hormone-specific responses
           [Computational Biology]
    • Authors: Kateřina Machačkova; Květoslava Mlčochova, Pavlo Potalitsyn, Kateřina Hankova, Ondřeȷ Socha, Miloš Buděšinsky, Anȷa Muždalo, Martin Lepšik, Michaela Černekova, Jelena Radosavlȷević, Milan Fabry, Katarina Mitrova, Martina Chrudinova, Jingȷing Lin, Yevgen Yurenko, Pavel Hobza, Irena Selicharova, Lenka Žakova, Jiři Jiraček
      Pages: 17371 - 17382
      Abstract: Information on how insulin and insulin-like growth factors 1 and 2 (IGF-1 and -2) activate insulin receptors (IR-A and -B) and the IGF-1 receptor (IGF-1R) is crucial for understanding the difference in the biological activities of these peptide hormones. Cryo-EM studies have revealed that insulin uses its binding sites 1 and 2 to interact with IR-A and have identified several critical residues in binding site 2. However, mutagenesis studies suggest that Ile-A10, Ser-A12, Leu-A13, and Glu-A17 also belong to insulin's site 2. Here, to resolve this discrepancy, we mutated these insulin residues and the equivalent residues in IGFs. Our findings revealed that equivalent mutations in the hormones can result in differential biological effects and that these effects can be receptor-specific. We noted that the insulin positions A10 and A17 are important for its binding to IR-A and IR-B and IGF-1R and that A13 is important only for IR-A and IR-B binding. The IGF-1/IGF-2 positions 51/50 and 54/53 did not appear to play critical roles in receptor binding, but mutations at IGF-1 position 58 and IGF-2 position 57 affected the binding. We propose that IGF-1 Glu-58 interacts with IGF-1R Arg-704 and belongs to IGF-1 site 1, a finding supported by the NMR structure of the less active Asp-58–IGF-1 variant. Computational analyses indicated that the aforementioned mutations can affect internal insulin dynamics and inhibit adoption of a receptor-bound conformation, important for binding to receptor site 1. We provide a molecular model and alternative hypotheses for how the mutated insulin residues affect activity.
      PubDate: 2019-11-15T00:06:23-08:00
      DOI: 10.1074/jbc.RA119.010072
      Issue No: Vol. 294, No. 46 (2019)
  • Intrinsic disorder and amino acid specificity modulate binding of the WW2
           domain in kidney and brain protein (KIBRA) to synaptopodin [Molecular
    • Authors: Ethiene Kwok; Diego J. Rodriguez, Joachim Kremerskothen, Afua Nyarko
      Pages: 17383 - 17394
      Abstract: The second WW domain (WW2) of the kidney and brain scaffolding protein, KIBRA, has an isoleucine (Ile-81) rather than a second conserved tryptophan and is primarily unstructured. However, it adopts the canonical triple-stranded antiparallel β-sheet structure of WW domains when bound to a two-PPXY motif peptide of the synaptic protein Dendrin. Here, using a series of biophysical experiments, we demonstrate that the WW2 domain remains largely disordered when bound to a 69-residue two-PPXY motif polypeptide of the synaptic and podocyte protein synaptopodin (SYNPO). Isothermal titration calorimetry and CD experiments revealed that the interactions of the disordered WW2 domain with SYNPO are significantly weaker than SYNPO's interactions with the well-folded WW1 domain and that an I81W substitution in the WW2 domain neither enhances binding affinity nor induces substantial WW2 domain folding. In the tandem polypeptide, the two WW domains synergized, enhancing the overall binding affinity with the I81W variant tandem polypeptide 2-fold compared with the WT polypeptide. Solution NMR results showed that SYNPO binding induces small but definite chemical shift perturbations in the WW2 domain, confirming the disordered state of the WW2 domain in this complex. These analyses also disclosed that SYNPO binds the tandem WW domain polypeptide in an antiparallel manner, that is, the WW1 domain binds the second PPXY motif of SYNPO. We propose a binding model consisting of a bipartite interaction mode in which the largely disordered WW2 forms a “fuzzy” complex with SYNPO. This binding mode may be important for specific cellular functions.
      PubDate: 2019-11-15T00:06:23-08:00
      DOI: 10.1074/jbc.RA119.009589
      Issue No: Vol. 294, No. 46 (2019)
  • Mechanically activated Piezo1 channels of cardiac fibroblasts stimulate
           p38 mitogen-activated protein kinase activity and interleukin-6 secretion
           [Signal Transduction]
    • Authors: Nicola M. Blythe; Katsuhiko Muraki, Melanie J. Ludlow, Vasili Stylianidis, Hamish T. J. Gilbert, Elizabeth L. Evans, Kevin Cuthbertson, Richard Foster, Joe Swift, Jing Li, Mark J. Drinkhill, Frans A. van Nieuwenhoven, Karen E. Porter, David J. Beech, Neil A. Turner
      Pages: 17395 - 17408
      Abstract: Piezo1 is a mechanosensitive cation channel with widespread physiological importance; however, its role in the heart is poorly understood. Cardiac fibroblasts help preserve myocardial integrity and play a key role in regulating its repair and remodeling following stress or injury. Here we investigated Piezo1 expression and function in cultured human and mouse cardiac fibroblasts. RT-PCR experiments confirmed that Piezo1 mRNA in cardiac fibroblasts is expressed at levels similar to those in endothelial cells. The results of a Fura-2 intracellular Ca2+ assay validated Piezo1 as a functional ion channel that is activated by its agonist, Yoda1. Yoda1-induced Ca2+ entry was inhibited by Piezo1 blockers (gadolinium and ruthenium red) and was reduced proportionally by siRNA-mediated Piezo1 knockdown or in murine Piezo1+/− cells. Results from cell-attached patch clamp recordings on human cardiac fibroblasts established that they contain mechanically activated ion channels and that their pressure responses are reduced by Piezo1 knockdown. Investigation of Yoda1 effects on selected remodeling genes indicated that Piezo1 activation increases both mRNA levels and protein secretion of IL-6, a pro-hypertrophic and profibrotic cytokine, in a Piezo1-dependent manner. Moreover, Piezo1 knockdown reduced basal IL-6 expression from cells cultured on softer collagen-coated substrates. Multiplex kinase activity profiling combined with kinase inhibitor experiments and phosphospecific immunoblotting established that Piezo1 activation stimulates IL-6 secretion via the p38 mitogen-activated protein kinase downstream of Ca2+ entry. In summary, cardiac fibroblasts express mechanically activated Piezo1 channels coupled to secretion of the paracrine signaling molecule IL-6. Piezo1 may therefore be important in regulating cardiac remodeling.
      PubDate: 2019-11-15T00:06:23-08:00
      DOI: 10.1074/jbc.RA119.009167
      Issue No: Vol. 294, No. 46 (2019)
  • Genetic code expansion and photocross-linking identify different
           {beta}-arrestin binding modes to the angiotensin II type 1 receptor
           [Signal Transduction]
    • Authors: Laurence Gagnon; Yubo Cao, Aaron Cho, Dana Sedki, Thomas Huber, Thomas P. Sakmar, Stephane A. Laporte
      Pages: 17409 - 17420
      Abstract: The angiotensin II (AngII) type 1 receptor (AT1R) is a member of the G protein–coupled receptor (GPCR) family and binds β-arrestins (β-arrs), which regulate AT1R signaling and trafficking. These processes can be biased by different ligands or mutations in the AGTR1 gene. As for many GPCRs, the exact details for AT1R–β-arr interactions driven by AngII or β-arr–biased ligands remain largely unknown. Here, we used the amber-suppression technology to site-specifically introduce the unnatural amino acid (UAA) p-azido-l-phenylalanine (azF) into the intracellular loops (ICLs) and the C-tail of AT1R. Our goal was to generate competent photoreactive receptors that can be cross-linked to β-arrs in cells. We performed UV-mediated photolysis of 25 different azF-labeled AT1Rs to cross-link β-arr1 to AngII-bound receptors, enabling us to map important contact sites in the C-tail and in the ICL2 and ICL3 of the receptor. The extent of AT1R–β-arr1 cross-linking among azF-labeled receptors differed, revealing variability in β-arr's contact mode with the different AT1R domains. Moreover, the signature of ligated AT1R–β-arr complexes from a subset of azF-labeled receptors also differed between AngII and β-arr–biased ligand stimulation of receptors and between azF-labeled AT1R bearing and that lacking a bias signaling mutation. These observations further implied distinct interaction modalities of the AT1R–β-arr1 complex in biased signaling conditions. Our findings demonstrate that this photocross-linking approach is useful for understanding GPCR–β-arr complexes in different activation states and could be extended to study other protein–protein interactions in cells.
      PubDate: 2019-11-15T00:06:23-08:00
      DOI: 10.1074/jbc.RA119.010324
      Issue No: Vol. 294, No. 46 (2019)
  • Comparative analysis of the catalytic regulation of NEDD4-1 and WWP2
           ubiquitin ligases [Protein Synthesis and Degradation]
    • Authors: Hanjie Jiang; Stefani N. Thomas, Zan Chen, Claire Y. Chiang, Philip A. Cole
      Pages: 17421 - 17436
      Abstract: NEDD4-1 E3 ubiquitin protein ligase (NEDD4-1) and WW domain-containing E3 ubiquitin ligase (WWP2) are HECT family ubiquitin E3 ligases. They catalyze Lys ubiquitination of themselves and other proteins and are important in cell growth and differentiation. Regulation of NEDD4-1 and WWP2 catalytic activities is important for controlling cellular protein homeostasis, and their dysregulation may lead to cancer and other diseases. Previous work has implicated noncatalytic regions, including the C2 domain and/or WW domain linkers in NEDD4-1 and WWP2, in contributing to autoinhibition of the catalytic HECT domains by intramolecular interactions. Here, we explored the molecular mechanisms of these NEDD4-1 and WWP2 regulatory regions and their interplay with allosteric binding proteins such as Nedd4 family-interacting protein (NDFIP1), engineered ubiquitin variants, and linker phosphomimics. We found that in addition to influencing catalytic activities, the WW domain linker regions in NEDD4-1 and WWP2 can impact product distribution, including the degree of polyubiquitination and Lys-48 versus Lys-63 linkages. We show that allosteric activation by NDFIP1 or engineered ubiquitin variants is largely mediated by relief of WW domain linker autoinhibition. WWP2-mediated ubiquitination of WW domain-binding protein 2 (WBP2), phosphatase and tensin homolog (PTEN), and p62 proteins by WWP2 suggests that substrate ubiquitination can also be influenced by WW linker autoinhibition, although to differing extents. Overall, our results provide a deeper understanding of the intricate and multifaceted set of regulatory mechanisms in the control of NEDD4-1–related ubiquitin ligases.
      PubDate: 2019-11-15T00:06:23-08:00
      DOI: 10.1074/jbc.RA119.009211
      Issue No: Vol. 294, No. 46 (2019)
  • Impact of key residues within chloroplast thioredoxin-f on recognition for
           reduction and oxidation of target proteins [Bioenergetics]
    • Authors: Yuichi Yokochi; Kazunori Sugiura, Kazuhiro Takemura, Keisuke Yoshida, Satoshi Hara, Ken-ichi Wakabayashi, Akio Kitao, Toru Hisabori
      Pages: 17437 - 17450
      Abstract: Thioredoxin (Trx) is a redox-responsive protein that modulates the activities of its target proteins mostly by reducing their disulfide bonds. In chloroplasts, five Trx isoforms (Trx-f, Trx-m, Trx-x, Trx-y, and Trx-z) regulate various photosynthesis-related enzymes with distinct target selectivity. To elucidate the determinants of the target selectivity of each Trx isoform, here we investigated the residues responsible for target recognition by Trx-f, the most well-studied chloroplast-resident Trx. As reported previously, we found that positively-charged residues on the Trx-f surface are involved in the interactions with its targets. Moreover, several residues that are specifically conserved in Trx-f (e.g. Cys-126 and Thr-158) were also involved in interactions with target proteins. The validity of these residues was examined by the molecular dynamics simulation. In addition, we validated the impact of these key residues on target protein reduction by studying (i) Trx-m variants into which we introduced the key residues for Trx-f and (ii) Trx-like proteins, named atypical Cys His-rich Trx 1 (ACHT1) and ACHT2a, that also contain these key residues. These artificial or natural protein variants could reduce Trx-f–specific targets, indicating that the key residues for Trx-f are critical for Trx-f–specific target recognition. Furthermore, we demonstrate that ACHT1 and ACHT2a efficiently oxidize some Trx-f–specific targets, suggesting that its target selectivity also contributes to the oxidative regulation process. Our results reveal the key residues for Trx-f–specific target recognition and uncover ACHT1 and ACHT2a as oxidation factors of their target proteins, providing critical insight into redox regulation of photosynthesis.
      PubDate: 2019-11-15T00:06:23-08:00
      DOI: 10.1074/jbc.RA119.010401
      Issue No: Vol. 294, No. 46 (2019)
  • Myosin motor domains carrying mutations implicated in early or late onset
           hypertrophic cardiomyopathy have similar properties [Molecular Biophysics]
    • Authors: Carlos D. Vera; Chloe A. Johnson, Jonathan Walklate, Arjun Adhikari, Marina Svicevic, Srboljub M. Mijailovich, Ariana C. Combs, Stephen J. Langer, Kathleen M. Ruppel, James A. Spudich, Michael A. Geeves, Leslie A. Leinwand
      Pages: 17451 - 17462
      Abstract: Hypertrophic cardiomyopathy (HCM) is a common genetic disorder characterized by left ventricular hypertrophy and cardiac hyper-contractility. Mutations in the β-cardiac myosin heavy chain gene (β-MyHC) are a major cause of HCM, but the specific mechanistic changes to myosin function that lead to this disease remain incompletely understood. Predicting the severity of any β-MyHC mutation is hindered by a lack of detailed examinations at the molecular level. Moreover, because HCM can take ≥20 years to develop, the severity of the mutations must be somewhat subtle. We hypothesized that mutations that result in early onset disease would have more severe changes in function than do later onset mutations. Here, we performed steady-state and transient kinetic analyses of myosins carrying one of seven missense mutations in the motor domain. Of these seven, four were previously identified in early onset cardiomyopathy screens. We used the parameters derived from these analyses to model the ATP-driven cross-bridge cycle. Contrary to our hypothesis, the results indicated no clear differences between early and late onset HCM mutations. Despite the lack of distinction between early and late onset HCM, the predicted occupancy of the force-holding actin·myosin·ADP complex at [Actin] = 3 Kapp along with the closely related duty ratio (the fraction of myosin in strongly attached force-holding states), and the measured ATPases all changed in parallel (in both sign and degree of change) compared with wildtype (WT) values. Six of the seven HCM mutations were clearly distinct from a set of previously characterized DCM mutations.
      PubDate: 2019-11-15T00:06:23-08:00
      DOI: 10.1074/jbc.RA119.010563
      Issue No: Vol. 294, No. 46 (2019)
  • Kinetic and structural evidence that Asp-678 plays multiple roles in
           catalysis by the quinoprotein glycine oxidase [Bioenergetics]
    • Authors: Kyle J. Mamounis; Dante Avalos, Erik T. Yukl, Victor L. Davidson
      Pages: 17463 - 17470
      Abstract: PlGoxA from Pseudoalteromonas luteoviolacea is a glycine oxidase that utilizes a protein-derived cysteine tryptophylquinone (CTQ) cofactor. A notable feature of its catalytic mechanism is that it forms a stable product-reduced CTQ adduct that is not hydrolyzed in the absence of O2. Asp-678 resides near the quinone moiety of PlGoxA, and an Asp is structurally conserved in this position in all tryptophylquinone enzymes. In those other enzymes, mutation of that Asp results in no or negligible CTQ formation. In this study, mutation of Asp-678 in PlGoxA did not abolish CTQ formation. This allowed, for the first time, studying the role of this residue in catalysis. D678A and D678N substitutions yielded enzyme variants with CTQ, which did not react with glycine, although glycine was present in the crystal structures in the active site. D678E PlGoxA was active but exhibited a much slower kcat. This mutation altered the kinetic mechanism of the reductive half-reaction such that one could observe a previously undetected reactive intermediate, an initial substrate-oxidized CTQ adduct, which converted to the product-reduced CTQ adduct. These results indicate that Asp-678 is involved in the initial deprotonation of the amino group of glycine, enabling nucleophilic attack of CTQ, as well as the deprotonation of the substrate-oxidized CTQ adduct, which is coupled to CTQ reduction. The structures also suggest that Asp-678 is acting as a proton relay that directs these protons to a water channel that connects the active sites on the subunits of this homotetrameric enzyme.
      PubDate: 2019-11-15T00:06:23-08:00
      DOI: 10.1074/jbc.RA119.011255
      Issue No: Vol. 294, No. 46 (2019)
  • A thiazole-derived oridonin analogue exhibits antitumor activity by
           directly and allosterically inhibiting STAT3 [Gene Regulation]
    • Authors: Xiaofei Shen; Lin Zhao, Peihao Chen, Yanqiu Gong, Dingdong Liu, Xia Zhang, Lunzhi Dai, Qingxiang Sun, Jizhong Lou, Zhong Jin, Baohua Zhang, Dawen Niu, Ceshi Chen, Xiangbing Qi, Da Jia
      Pages: 17471 - 17486
      Abstract: Constitutive activation of signal transducer and activator of transcription 3 (STAT3) occurs in ∼70% of human cancers, and STAT3 is regarded as one of the most promising targets for cancer therapy. However, specific direct STAT3 inhibitors remain to be developed. Oridonin is an ent-kaurane plant-derived diterpenoid with anti-cancer and anti-inflammatory activities. Here, using an array of cell-based and biochemical approaches, including cell proliferation and apoptosis assays, pulldown and reporter gene assays, site-directed mutagenesis, and molecular dynamics analyses, we report that a thiazole-derived oridonin analogue, CYD0618, potently and directly inhibits STAT3. We found that CYD0618 covalently binds to Cys-542 in STAT3 and suppresses its activity through an allosteric effect, effectively reducing STAT3 dimerization and nuclear translocation, as well as decreasing expression of STAT3-targeted oncogenes. Remarkably, CYD0618 not only strongly inhibited growth of multiple cancer cell lines that harbor constitutive STAT3 activation, but it also suppressed in vivo tumor growth via STAT3 inhibition. Taken together, our findings suggest Cys-542 as a druggable site for selectively inhibiting STAT3 and indicate that CYD0618 represents a promising lead compound for developing therapeutic agents against STAT3-driven diseases.
      PubDate: 2019-11-15T00:06:23-08:00
      DOI: 10.1074/jbc.RA119.009801
      Issue No: Vol. 294, No. 46 (2019)
  • The transcription factor PU.1 mediates enhancer-promoter looping that is
           required for IL-1{beta} eRNA and mRNA transcription in mouse melanoma and
           macrophage cell lines [Immunology]
    • Authors: Soon-Duck Ha; Woohyun Cho, Rodney P. DeKoter, Sung Ouk Kim
      Pages: 17487 - 17500
      Abstract: The DNA-binding protein PU.1 is a myeloid lineage–determining and pioneering transcription factor due to its ability to bind “closed” genomic sites and maintain “open” chromatin state for myeloid lineage–specific genes. The precise mechanism of PU.1 in cell type–specific programming is yet to be elucidated. The melanoma cell line B16BL6, although it is nonmyeloid lineage, expressed Toll-like receptors and activated the transcription factor NF-κB upon stimulation by the bacterial cell wall component lipopolysaccharide. However, it did not produce cytokines, such as IL-1β mRNA. Ectopic PU.1 expression induced remodeling of a novel distal enhancer (located ∼10 kbp upstream of the IL-1β transcription start site), marked by nucleosome depletion, enhancer-promoter looping, and histone H3 lysine 27 acetylation (H3K27ac). PU.1 induced enhancer-promoter looping and H3K27ac through two distinct PU.1 regions. These PU.1-dependent events were independently required for subsequent signal-dependent and co-dependent events: NF-κB recruitment and further H3K27ac, both of which were required for enhancer RNA (eRNA) transcription. In murine macrophage RAW264.7 cells, these PU.1-dependent events were constitutively established and readily expressed eRNA and subsequently IL-1β mRNA by lipopolysaccharide stimulation. In summary, this study showed a sequence of epigenetic events in programming IL-1β transcription by the distal enhancer priming and eRNA production mediated by PU.1 and the signal-dependent transcription factor NF-κB.
      PubDate: 2019-11-15T00:06:23-08:00
      DOI: 10.1074/jbc.RA119.010149
      Issue No: Vol. 294, No. 46 (2019)
  • Xenogeneic modulation of the ClpCP protease of Bacillus subtilis by a
           phage-encoded adaptor-like protein [Protein Synthesis and Degradation]
    • Authors: Nancy Mulvenna; Ingo Hantke, Lynn Burchell, Sophie Nicod, David Bell, Kurşad Turgay, Sivaramesh Wigneshweraraȷ
      Pages: 17501 - 17511
      Abstract: Like eukaryotic and archaeal viruses, which coopt the host's cellular pathways for their replication, bacteriophages have evolved strategies to alter the metabolism of their bacterial host. SPO1 bacteriophage infection of Bacillus subtilis results in comprehensive remodeling of cellular processes, leading to conversion of the bacterial cell into a factory for phage progeny production. A cluster of 26 genes in the SPO1 genome, called the host takeover module, encodes for potentially cytotoxic proteins that specifically shut down various processes in the bacterial host, including transcription, DNA synthesis, and cell division. However, the properties and bacterial targets of many genes of the SPO1 host takeover module remain elusive. Through a systematic analysis of gene products encoded by the SPO1 host takeover module, here we identified eight gene products that attenuated B. subtilis growth. Of the eight phage gene products that attenuated bacterial growth, a 25-kDa protein called Gp53 was shown to interact with the AAA+ chaperone protein ClpC of the ClpCP protease of B. subtilis. Our results further reveal that Gp53 is a phage-encoded adaptor-like protein that modulates the activity of the ClpCP protease to enable efficient SPO1 phage progeny development. In summary, our findings indicate that the bacterial ClpCP protease is the target of xenogeneic (dys)regulation by a SPO1 phage–derived factor and add Gp53 to the list of antibacterial products that target bacterial protein degradation and therefore may have utility for the development of novel antibacterial agents.
      PubDate: 2019-11-15T00:06:23-08:00
      DOI: 10.1074/jbc.RA119.010007
      Issue No: Vol. 294, No. 46 (2019)
  • A piperidinol-containing molecule is active against Mycobacterium
           tuberculosis by inhibiting the mycolic acid flippase activity of MmpL3
    • Authors: Christian Dupont; Yushu Chen, Zhuȷun Xu, Francoise Roquet–Baneres, Mickael Blaise, Anne–Kathrin Witt, Faustine Dubar, Christophe Biot, Yann Guerardel, Florian P. Maurer, Shu–Sin Chng, Laurent Kremer
      Pages: 17512 - 17523
      Abstract: Mycobacterium tuberculosis, the causative agent of tuberculosis, remains a major human pathogen, and current treatment options to combat this disease are under threat because of the emergence of multidrug-resistant and extensively drug-resistant tuberculosis. High-throughput whole-cell screening of an extensive compound library has recently identified a piperidinol-containing molecule, PIPD1, as a potent lead compound against M. tuberculosis. Herein, we show that PIPD1 and related analogs exert in vitro bactericidal activity against the M. tuberculosis strain mc26230 and also against a panel of multidrug-resistant and extensively drug-resistant clinical isolates of M. tuberculosis, suggesting that PIPD1's mode of action differs from those of most first- and second-line anti-tubercular drugs. Selection and DNA sequencing of PIPD1-resistant mycobacterial mutants revealed the presence of single-nucleotide polymorphisms in mmpL3, encoding an inner membrane–associated mycolic acid flippase in M. tuberculosis. Results from functional assays with spheroplasts derived from a M. smegmatis strain lacking the endogenous mmpL3 gene but harboring the M. tuberculosis mmpL3 homolog indicated that PIPD1 inhibits the MmpL3-driven translocation of trehalose monomycolate across the inner membrane without altering the proton motive force. Using a predictive structural model of MmpL3 from M. tuberculosis, docking studies revealed a PIPD1-binding cavity recently found to accommodate different inhibitors in M. smegmatis MmpL3. In conclusion, our findings have uncovered bactericidal activity of a new chemical scaffold. Its anti-tubercular activity is mediated by direct inhibition of the flippase activity of MmpL3 rather than by inhibition of the inner membrane proton motive force, significantly advancing our understanding of MmpL3-targeted inhibition in mycobacteria.
      PubDate: 2019-11-15T00:06:23-08:00
      DOI: 10.1074/jbc.RA119.010135
      Issue No: Vol. 294, No. 46 (2019)
  • {beta}-Catenin mutations as determinants of hepatoblastoma phenotypes in
           mice [Metabolism]
    • Authors: Weiqi Zhang; Jennifer Meyfeldt, Huabo Wang, Sucheta Kulkarni, Jie Lu, Jordan A. Mandel, Brady Marburger, Ying Liu, Joanna E. Gorka, Sarangarajan Ranganathan, Edward V. Prochownik
      Pages: 17524 - 17542
      Abstract: Hepatoblastoma (HB) is the most common pediatric liver cancer. Although long-term survival of HB is generally favorable, it depends on clinical stage, tumor histology, and a variety of biochemical and molecular features. HB appears almost exclusively before the age of 3 years, is represented by seven histological subtypes, and is usually associated with highly heterogeneous somatic mutations in the catenin β1 (CTNNB1) gene, which encodes β-catenin, a Wnt ligand–responsive transcriptional co-factor. Numerous recurring β-catenin mutations, not previously documented in HB, have also been identified in various other pediatric and adult cancer types. Little is known about the underlying factors that determine the above HB features and behaviors or whether non-HB–associated β-catenin mutations are tumorigenic when expressed in hepatocytes. Here, we investigated the oncogenic properties of 14 different HB– and non-HB–associated β-catenin mutants encoded by Sleeping Beauty vectors following their delivery into the mouse liver by hydrodynamic tail-vein injection. We show that all β-catenin mutations, as well as WT β-catenin, are tumorigenic when co-expressed with a mutant form of yes-associated protein (YAP). However, tumor growth rates, histologies, nuclear-to-cytoplasmic partitioning, and metabolic and transcriptional landscapes were strongly influenced by the identities of the β-catenin mutations. These findings provide a context for understanding at the molecular level the notable biological diversity of HB.
      PubDate: 2019-11-15T00:06:23-08:00
      DOI: 10.1074/jbc.RA119.009979
      Issue No: Vol. 294, No. 46 (2019)
  • Calmodulin is involved in the dual subcellular location of two chloroplast
           proteins [Cell Biology]
    • Authors: Lucas Moyet; Daniel Salvi, Imen Bouchnak, Stephane Miras, Laura Perrot, Daphne Seigneurin–Berny, Marcel Kuntz, Norbert Rolland
      Pages: 17543 - 17554
      Abstract: Cell compartmentalization is an essential process by which eukaryotic cells separate and control biological processes. Although calmodulins are well-known to regulate catalytic properties of their targets, we show here their involvement in the subcellular location of two plant proteins. Both proteins exhibit a dual location, namely in the cytosol in addition to their association to plastids (where they are known to fulfil their role). One of these proteins, ceQORH, a long-chain fatty acid reductase, was analyzed in more detail, and its calmodulin-binding site was identified by specific mutations. Such a mutated form is predominantly targeted to plastids at the expense of its cytosolic location. The second protein, TIC32, was also shown to be dependent on its calmodulin-binding site for retention in the cytosol. Complementary approaches (bimolecular fluorescence complementation and reverse genetics) demonstrated that the calmodulin isoform CAM5 is specifically involved in the retention of ceQORH in the cytosol. This study identifies a new role for calmodulin and sheds new light on the intriguing CaM-binding properties of hundreds of plastid proteins, despite the fact that no CaM or CaM-like proteins were identified in plastids.
      PubDate: 2019-11-15T00:06:23-08:00
      DOI: 10.1074/jbc.RA119.010846
      Issue No: Vol. 294, No. 46 (2019)
  • FOXO1 transcription factor regulates chondrogenic differentiation through
           transforming growth factor {beta}1 signaling [Developmental Biology]
    • Authors: Ichiro Kurakazu; Yukio Akasaki, Mitsumasa Hayashida, Hidetoshi Tsushima, Norio Goto, Takuya Sueishi, Masakazu Toya, Masanari Kuwahara, Ken Okazaki, Tomas Duffy, Martin K. Lotz, Yasuharu Nakashima
      Pages: 17555 - 17569
      Abstract: The forkhead box O (FOXO) proteins are transcription factors involved in the differentiation of many cell types. Type II collagen (Col2) Cre-Foxo1-knockout and Col2-Cre-Foxo1,3,4 triple-knockout mice exhibit growth plate malformation. Moreover, recent studies have reported that in some cells, the expressions and activities of FOXOs are promoted by transforming growth factor β1 (TGFβ1), a growth factor playing a key role in chondrogenic differentiation. Here, using a murine chondrogenic cell line (ATDC5), mouse embryos, and human mesenchymal stem cells, we report the mechanisms by which FOXOs affect chondrogenic differentiation. FOXO1 expression increased along with chondrogenic differentiation, and FOXO1 inhibition suppressed chondrogenic differentiation. TGFβ1/SMAD signaling promoted expression and activity of FOXO1. In ATDC5, FOXO1 knockdown suppressed expression of sex-determining region Y box 9 (Sox9), a master regulator of chondrogenic differentiation, resulting in decreased collagen type II α1 (Col2a1) and aggrecan (Acan) expression after TGFβ1 treatment. On the other hand, chemical FOXO1 inhibition suppressed Col2a1 and Acan expression without suppressing Sox9. To investigate the effects of FOXO1 on chondrogenic differentiation independently of SOX9, we examined FOXO1's effects on the cell cycle. FOXO1 inhibition suppressed expression of p21 and cell-cycle arrest in G0/G1 phase. Conversely, FOXO1 overexpression promoted expression of p21 and cell-cycle arrest. FOXO1 inhibition suppressed expression of nascent p21 RNA by TGFβ1, and FOXO1 bound the p21 promoter. p21 inhibition suppressed expression of Col2a1 and Acan during chondrogenic differentiation. These results suggest that FOXO1 is necessary for not only SOX9 expression, but also cell-cycle arrest during chondrogenic differentiation via TGFβ1 signaling.
      PubDate: 2019-11-15T00:06:23-08:00
      DOI: 10.1074/jbc.RA119.009409
      Issue No: Vol. 294, No. 46 (2019)
  • Proteomic analysis of affinity-purified 26S proteasomes identifies a suite
           of assembly chaperones in Arabidopsis [Plant Biology]
    • Authors: David C. Gemperline; Richard S. Marshall, Kwang-Hee Lee, Qingzhen Zhao, Weiming Hu, Fionn McLoughlin, Mark Scalf, Lloyd M. Smith, Richard D. Vierstra
      Pages: 17570 - 17592
      Abstract: The 26S proteasome is an essential protease that selectively eliminates dysfunctional and short-lived regulatory proteins in eukaryotes. To define the composition of this proteolytic machine in plants, we tagged either the core protease (CP) or the regulatory particle (RP) sub-complexes in Arabidopsis to enable rapid affinity purification followed by mass spectrometric analysis. Studies on proteasomes enriched from whole seedlings, with or without ATP needed to maintain the holo-proteasome complex, identified all known proteasome subunits but failed to detect isoform preferences, suggesting that Arabidopsis does not construct distinct proteasome sub-types. We also detected a suite of proteasome-interacting proteins, including likely orthologs of the yeast and mammalian chaperones Pba1, Pba2, Pba3, and Pba4 that assist in CP assembly; Ump1 that helps connect CP half-barrels; Nas2, Nas6, and Hsm3 that assist in RP assembly; and Ecm29 that promotes CP–RP association. Proteasomes from seedlings exposed to the proteasome inhibitor MG132 accumulated assembly intermediates, reflecting partially built proteasome sub-complexes associated with assembly chaperones, and the CP capped with the PA200/Blm10 regulator. Genetic analyses of Arabidopsis UMP1 revealed that, unlike in yeast, this chaperone is essential, with mutants lacking the major UMP1a and UMP1b isoforms displaying a strong gametophytic defect. Single ump1 mutants were hypersensitive to conditions that induce proteotoxic, salt and osmotic stress, and also accumulated several proteasome assembly intermediates, consistent with its importance for CP construction. Insights into the chaperones reported here should enable study of the assembly events that generate the 26S holo-proteasome in Arabidopsis from the collection of 64 or more subunits.
      PubDate: 2019-11-15T00:06:23-08:00
      DOI: 10.1074/jbc.RA119.010219
      Issue No: Vol. 294, No. 46 (2019)
  • Mitochondrial amidoxime-reducing component 2 (MARC2) has a significant
           role in N-reductive activity and energy metabolism [Enzymology]
    • Authors: Sophia Rixen; Antje Havemeyer, Anita Tyl-Bielicka, Kazimiera Pysniak, Marta Gajewska, Maria Kulecka, Jerzy Ostrowski, Michal Mikula, Bernd Clement
      Pages: 17593 - 17602
      Abstract: The mitochondrial amidoxime-reducing component (MARC) is a mammalian molybdenum-containing enzyme. All annotated mammalian genomes harbor two MARC genes, MARC1 and MARC2, which share a high degree of sequence similarity. Both molybdoenzymes reduce a variety of N-hydroxylated compounds. Besides their role in N-reductive drug metabolism, only little is known about their physiological functions. In this study, we characterized an existing KO mouse model lacking the functional MARC2 gene and fed a high-fat diet and also performed in vivo and in vitro experiments to characterize reductase activity toward known MARC substrates. MARC2 KO significantly decreased reductase activity toward several N-oxygenated substrates, and for typical MARC substrates, only small residual reductive activity was still detectable in MARC2 KO mice. The residual detected reductase activity in MARC2 KO mice could be explained by MARC1 expression that was hardly unaffected by KO, and we found no evidence of significant activity of other reductase enzymes. These results clearly indicate that MARC2 is mainly responsible for N-reductive biotransformation in mice. Striking phenotypical features of MARC2 KO mice were lower body weight, increased body temperature, decreased levels of total cholesterol, and increased glucose levels, supporting previous findings that MARC2 affects energy pathways. Of note, the MARC2 KO mice were resistant to high-fat diet–induced obesity. We propose that the MARC2 KO mouse model could be a powerful tool for predicting MARC-mediated drug metabolism and further investigating MARC's roles in energy homeostasis.
      PubDate: 2019-11-15T00:06:23-08:00
      DOI: 10.1074/jbc.RA119.007606
      Issue No: Vol. 294, No. 46 (2019)
  • Heparin potentiates Avastin-mediated inhibition of VEGF binding to
           fibronectin and rescues Avastin activity at acidic pH [Cell Biology]
    • Authors: Divyabharathy Tsiros; Casey E. Sheehy, Surenna Pecchia, Matthew A. Nugent
      Pages: 17603 - 17611
      Abstract: Vascular endothelial growth factor-A (VEGF) plays a critical role in stimulating angiogenesis in normal and disease states. Anti-VEGF antibodies have been developed to manage pathological angiogenesis. Bevacizumab, sold under the brand name Avastin, is a humanized mAb that binds VEGF and blocks its binding to its signaling receptor, VEGF receptor 2, and is used to treat patients with a variety of cancers or retinal disorders. The ability of Avastin to modulate other nonreceptor interactions of VEGF has not been fully defined. In this study, we investigated Avastin's capacity to modulate VEGF165 binding to porcine aortic endothelial cells and to heparin and fibronectin (FN) across a range of pH values (pH 5–8). We observed that Avastin slightly enhanced VEGF binding to heparin and that heparin increased VEGF binding to Avastin. In contrast, Avastin inhibited VEGF binding to cells and FN, yet Avastin could still bind to VEGF that was bound to FN, indicating that these binding events are not mutually exclusive. Avastin binding to VEGF was dramatically reduced at acidic pH values (pH 5.0–6.5), whereas VEGF binding to FN and nonreceptor sites on cells was enhanced. Interestingly, the reduced Avastin–VEGF binding at acidic pH was rescued by heparin, as was Avastin's ability to inhibit VEGF binding to cells. These results suggest that heparin might be used to expand the clinical utility of Avastin. Our findings highlight the importance of defining the range of VEGF interactions to fully predict antibody activity within a complex biological setting.
      PubDate: 2019-11-15T00:06:23-08:00
      DOI: 10.1074/jbc.RA119.009194
      Issue No: Vol. 294, No. 46 (2019)
  • A dual-chain assembly pathway generates the high structural diversity of
           cell-wall polysaccharides in Lactococcus lactis [Glycobiology and
           Extracellular Matrices]
    • Authors: Ilias Theodorou; Pascal Courtin, Simon Palussiere, Saulius Kulakauskas, Elena Bidnenko, Christine Pechoux, Francois Fenaille, Christophe Penno, Jennifer Mahony, Douwe van Sinderen, Marie–Pierre Chapot–Chartier
      Pages: 17612 - 17625
      Abstract: In Lactococcus lactis, cell-wall polysaccharides (CWPSs) act as receptors for many bacteriophages, and their structural diversity among strains explains, at least partially, the narrow host range of these viral predators. Previous studies have reported that lactococcal CWPS consists of two distinct components, a variable chain exposed at the bacterial surface, named polysaccharide pellicle (PSP), and a more conserved rhamnan chain anchored to, and embedded inside, peptidoglycan. These two chains appear to be covalently linked to form a large heteropolysaccharide. The molecular machinery for biosynthesis of both components is encoded by a large gene cluster, named cwps. In this study, using a CRISPR/Cas-based method, we performed a mutational analysis of the cwps genes. MALDI-TOF MS-based structural analysis of the mutant CWPS combined with sequence homology, transmission EM, and phage sensitivity analyses enabled us to infer a role for each protein encoded by the cwps cluster. We propose a comprehensive CWPS biosynthesis scheme in which the rhamnan and PSP chains are independently synthesized from two distinct lipid-sugar precursors and are joined at the extracellular side of the cytoplasmic membrane by a mechanism involving a membrane-embedded glycosyltransferase with a GT-C fold. The proposed scheme encompasses a system that allows extracytoplasmic modification of rhamnan by complex substituting oligo-/polysaccharides. It accounts for the extensive diversity of CWPS structures observed among lactococci and may also have relevance to the biosynthesis of complex rhamnose-containing CWPSs in other Gram-positive bacteria.
      PubDate: 2019-11-15T00:06:23-08:00
      DOI: 10.1074/jbc.RA119.009957
      Issue No: Vol. 294, No. 46 (2019)
  • Manganese co-localizes with calcium and phosphorus in Chlamydomonas
           acidocalcisomes and is mobilized in manganese-deficient conditions [Plant
    • Authors: Munkhtsetseg Tsednee; Madeli Castruita, Patrice A. Salome, Aȷay Sharma, Brianne E. Lewis, Stefan R. Schmollinger, Daniela Strenkert, Kristen Holbrook, Marisa S. Otegui, Kaustav Khatua, Sayani Das, Ankona Datta, Si Chen, Christina Ramon, Martina Ralle, Peter K. Weber, Timothy L. Stemmler, Jennifer Pett–Ridge, Brian M. Hoffman, Sabeeha S. Merchant
      Pages: 17626 - 17641
      Abstract: Exposing cells to excess metal concentrations well beyond the cellular quota is a powerful tool for understanding the molecular mechanisms of metal homeostasis. Such improved understanding may enable bioengineering of organisms with improved nutrition and bioremediation capacity. We report here that Chlamydomonas reinhardtii can accumulate manganese (Mn) in proportion to extracellular supply, up to 30-fold greater than its typical quota and with remarkable tolerance. As visualized by X-ray fluorescence microscopy and nanoscale secondary ion MS (nanoSIMS), Mn largely co-localizes with phosphorus (P) and calcium (Ca), consistent with the Mn-accumulating site being an acidic vacuole, known as the acidocalcisome. Vacuolar Mn stores are accessible reserves that can be mobilized in Mn-deficient conditions to support algal growth. We noted that Mn accumulation depends on cellular polyphosphate (polyP) content, indicated by 1) a consistent failure of C. reinhardtii vtc1 mutant strains, which are deficient in polyphosphate synthesis, to accumulate Mn and 2) a drastic reduction of the Mn storage capacity in P-deficient cells. Rather surprisingly, X-ray absorption spectroscopy, EPR, and electron nuclear double resonance revealed that only little Mn2+ is stably complexed with polyP, indicating that polyP is not the final Mn ligand. We propose that polyPs are a critical component of Mn accumulation in Chlamydomonas by driving Mn relocation from the cytosol to acidocalcisomes. Within these structures, polyP may, in turn, escort vacuolar Mn to a number of storage ligands, including phosphate and phytate, and other, yet unidentified, compounds.
      PubDate: 2019-11-15T00:06:23-08:00
      DOI: 10.1074/jbc.RA119.009130
      Issue No: Vol. 294, No. 46 (2019)
  • Functionally critical residues in the aminoglycoside resistance-associated
           methyltransferase RmtC play distinct roles in 30S substrate recognition
    • Authors: Meisam Nosrati; Debayan Dey, Atousa Mehrani, Sarah E. Strassler, Natalia Zelinskaya, Eric D. Hoffer, Scott M. Stagg, Christine M. Dunham, Graeme L. Conn
      Pages: 17642 - 17653
      Abstract: Methylation of the small ribosome subunit rRNA in the ribosomal decoding center results in exceptionally high-level aminoglycoside resistance in bacteria. Enzymes that methylate 16S rRNA on N7 of nucleotide G1405 (m7G1405) have been identified in both aminoglycoside-producing and clinically drug-resistant pathogenic bacteria. Using a fluorescence polarization 30S-binding assay and a new crystal structure of the methyltransferase RmtC at 3.14 Å resolution, here we report a structure-guided functional study of 30S substrate recognition by the aminoglycoside resistance-associated 16S rRNA (m7G1405) methyltransferases. We found that the binding site for these enzymes in the 30S subunit directly overlaps with that of a second family of aminoglycoside resistance-associated 16S rRNA (m1A1408) methyltransferases, suggesting that both groups of enzymes may exploit the same conserved rRNA tertiary surface for docking to the 30S. Within RmtC, we defined an N-terminal domain surface, comprising basic residues from both the N1 and N2 subdomains, that directly contributes to 30S-binding affinity. In contrast, additional residues lining a contiguous adjacent surface on the C-terminal domain were critical for 16S rRNA modification but did not directly contribute to the binding affinity. The results from our experiments define the critical features of m7G1405 methyltransferase–substrate recognition and distinguish at least two distinct, functionally critical contributions of the tested enzyme residues: 30S-binding affinity and stabilizing a binding-induced 16S rRNA conformation necessary for G1405 modification. Our study sets the scene for future high-resolution structural studies of the 30S-methyltransferase complex and for potential exploitation of unique aspects of substrate recognition in future therapeutic strategies.
      PubDate: 2019-11-15T00:06:23-08:00
      DOI: 10.1074/jbc.RA119.011181
      Issue No: Vol. 294, No. 46 (2019)
  • Physiologically relevant orthogonal assays for the discovery of
           small-molecule modulators of WIP1 phosphatase in high-throughput screens
    • Authors: Victor Clausse; Dingyin Tao, Subrata Debnath, Yuhong Fang, Harichandra D. Tagad, Yuhong Wang, Hongmao Sun, Christopher A. LeClair, Sharlyn J. Mazur, Kelly Lane, Zhen-Dan Shi, Olga Vasalatiy, Rebecca Eells, Lynn K. Baker, Mark J. Henderson, Martin R. Webb, Min Shen, Matthew D. Hall, Ettore Appella, Daniel H. Appella, Nathan P. Coussens
      Pages: 17654 - 17668
      Abstract: WT P53-Induced Phosphatase 1 (WIP1) is a member of the magnesium-dependent serine/threonine protein phosphatase (PPM) family and is induced by P53 in response to DNA damage. In several human cancers, the WIP1 protein is overexpressed, which is generally associated with a worse prognosis. Although WIP1 is an attractive therapeutic target, no potent, selective, and bioactive small-molecule modulator with favorable pharmacokinetics has been reported. Phosphatase enzymes are among the most challenging targets for small molecules because of the difficulty of achieving both modulator selectivity and bioavailability. Another major obstacle has been the availability of robust and physiologically relevant phosphatase assays that are suitable for high-throughput screening. Here, we describe orthogonal biochemical WIP1 activity assays that utilize phosphopeptides from native WIP1 substrates. We optimized an MS assay to quantify the enzymatically dephosphorylated peptide reaction product in a 384-well format. Additionally, a red-shifted fluorescence assay was optimized in a 1,536-well format to enable real-time WIP1 activity measurements through the detection of the orthogonal reaction product, Pi. We validated these two optimized assays by quantitative high-throughput screening against the National Center for Advancing Translational Sciences (NCATS) Pharmaceutical Collection and used secondary assays to confirm and evaluate inhibitors identified in the primary screen. Five inhibitors were further tested with an orthogonal WIP1 activity assay and surface plasmon resonance binding studies. Our results validate the application of miniaturized physiologically relevant and orthogonal WIP1 activity assays to discover small-molecule modulators from high-throughput screens.
      PubDate: 2019-11-15T00:06:23-08:00
      DOI: 10.1074/jbc.RA119.010201
      Issue No: Vol. 294, No. 46 (2019)
  • Hypoxia-inducible gene domain 1 proteins in yeast mitochondria protect
           against proton leak through complex IV [Membrane Biology]
    • Authors: Ngoc H. Hoang; Vera Strogolova, Jaramys J. Mosley, Rosemary A. Stuart, Jonathan Hosler
      Pages: 17669 - 17677
      Abstract: Hypoxia-inducible gene domain 1 (HIGD1) proteins are small integral membrane proteins, conserved from bacteria to humans, that associate with oxidative phosphorylation supercomplexes. Using yeast as a model organism, we have shown previously that its two HIGD1 proteins, Rcf1 and Rcf2, are required for the generation and maintenance of a normal membrane potential (ΔΨ) across the inner mitochondrial membrane (IMM). We postulated that the lower ΔΨ observed in the absence of the HIGD1 proteins may be due to decreased proton pumping by complex IV (CIV) or enhanced leak of protons across the IMM. Here we measured the ΔΨ generated by complex III (CIII) to discriminate between these possibilities. First, we found that the decreased ΔΨ observed in the absence of the HIGD1 proteins cannot be due to decreased proton pumping by CIV because CIII, operating alone, also exhibited a decreased ΔΨ when HIGD1 proteins were absent. Because CIII can neither lower its pumping stoichiometry nor transfer protons completely across the IMM, this result indicates that HIGD1 protein ablation enhances proton leak across the IMM. Second, we demonstrate that this proton leak occurs through CIV because ΔΨ generation by CIII is restored when CIV is removed from the cell. Third, the proton leak appeared to take place through an inactive population of CIV that accumulates when HIGD1 proteins are absent. We conclude that HIGD1 proteins in yeast prevent CIV inactivation, likely by preventing the loss of lipids bound within the Cox3 protein of CIV.
      PubDate: 2019-11-15T00:06:23-08:00
      DOI: 10.1074/jbc.RA119.010317
      Issue No: Vol. 294, No. 46 (2019)
  • How organic chemistry became one of UCLA's most popular classes [ASBMB
           Award Articles]
    • Authors: Neil K. Garg
      Pages: 17678 - 17683
      Abstract: Organic chemistry has a bad reputation, despite having a tremendous impact on our everyday lives. It has remained a notorious “weed-out” class for decades—striking fear in the hearts of students—and has long been viewed as a gatekeeper course for those interested in pursuing a career in medicine or other health-related professions. This personal account examines the underlying teaching philosophies that transformed organic chemistry into one of the most popular classes on the UCLA campus. Special emphasis is placed on ways to increase engagement and help students feel connected. Educational initiatives, including organic chemistry music videos and various online resources created in partnerships with students, will be discussed. It is hoped that this account will stimulate ideas that transcend scientific disciplines all for the benefit of student education.
      PubDate: 2019-11-15T00:06:23-08:00
      DOI: 10.1074/jbc.AW119.008141
      Issue No: Vol. 294, No. 46 (2019)
  • The biosynthetic diversity of the animal world [Enzymology]
    • Authors: Joshua P. Torres; Eric W. Schmidt
      Pages: 17684 - 17692
      Abstract: Secondary metabolites are often considered within the remit of bacterial or plant research, but animals also contain a plethora of these molecules with important functional roles. Classical feeding studies demonstrate that, whereas some are derived from diet, many of these compounds are made within the animals. In the past 15 years, the genetic and biochemical origin of several animal natural products has been traced to partnerships with symbiotic bacteria. More recently, a number of animal genome-encoded pathways to microbe-like natural products have come to light. These pathways are sometimes horizontally acquired from bacteria, but more commonly they unveil a new and diverse animal biochemistry. In this review, we highlight recent examples of characterized animal biosynthetic enzymes that reveal an unanticipated breadth and intricacy in animal secondary metabolism. The results so far suggest that there may be an immense diversity of animal small molecules and biosynthetic enzymes awaiting discovery. This biosynthetic dark matter is just beginning to be understood, providing a relatively untapped frontier for discovery.
      PubDate: 2019-11-15T00:06:23-08:00
      DOI: 10.1074/jbc.REV119.006130
      Issue No: Vol. 294, No. 46 (2019)
  • Control of cellular responses to mechanical cues through YAP/TAZ
           regulation [Cell Biology]
    • Authors: Ishani Dasgupta; Dannel McCollum
      Pages: 17693 - 17706
      Abstract: To perceive their three-dimensional environment, cells and tissues must be able to sense and interpret various physical forces like shear, tensile, and compression stress. These forces can be generated both internally and externally in response to physical properties, like substrate stiffness, cell contractility, and forces generated by adjacent cells. Mechanical cues have important roles in cell fate decisions regarding proliferation, survival, and differentiation as well as the processes of tissue regeneration and wound repair. Aberrant remodeling of the extracellular space and/or defects in properly responding to mechanical cues likely contributes to various disease states, such as fibrosis, muscle diseases, and cancer. Mechanotransduction involves the sensing and translation of mechanical forces into biochemical signals, like activation of specific genes and signaling cascades that enable cells to adapt to their physical environment. The signaling pathways involved in mechanical signaling are highly complex, but numerous studies have highlighted a central role for the Hippo pathway and other signaling networks in regulating the YAP and TAZ (YAP/TAZ) proteins to mediate the effects of mechanical stimuli on cellular behavior. How mechanical cues control YAP/TAZ has been poorly understood. However, rapid progress in the last few years is beginning to reveal a surprisingly diverse set of pathways for controlling YAP/TAZ. In this review, we will focus on how mechanical perturbations are sensed through changes in the actin cytoskeleton and mechanosensors at focal adhesions, adherens junctions, and the nuclear envelope to regulate YAP/TAZ.
      PubDate: 2019-11-15T00:06:23-08:00
      DOI: 10.1074/jbc.REV119.007963
      Issue No: Vol. 294, No. 46 (2019)
  • Correction: Engineered protein disaggregases mitigate toxicity of aberrant
           prion-like fusion proteins underlying sarcoma. [Additions and Corrections]
    • Authors: Jeremy J. Ryan; Macy L. Sprunger, Kayla Holthaus, James Shorter, Meredith E. Jackrel
      Pages: 17707 - 17707
      Abstract: VOLUME 294 (2019) PAGES 11286–11296Throughout the text, the abbreviations “EWS-FLI1” and “EWS-FLI3” refer to EWS-FLI1, type 1 and EWS-FLI1, type 3, respectively. These are EWS-FLI1 fusion proteins that arise from slightly different chromosomal translocations that are found naturally in human sarcomas (1). Domain maps for these distinct fusion proteins are shown in Fig. 2A.
      PubDate: 2019-11-15T00:06:23-08:00
      DOI: 10.1074/jbc.AAC119.011523
      Issue No: Vol. 294, No. 46 (2019)
  • Expression of Concern: Tumor suppressor SMAR1 activates and stabilizes p53
           through its arginine-serine-rich motif. [Expressions of Concern]
    • Pages: 17708 - 17708
      Abstract: VOLUME 280 (2005) PAGES 16019–16029The publisher of the Journal of Biological Chemistry is issuing an Expression of Concern to inform readers that credible concerns have been raised regarding some of the data and conclusions in the article listed above. The Journal of Biological Chemistry will provide additional information as it becomes available.
      PubDate: 2019-11-15T00:06:23-08:00
      DOI: 10.1074/jbc.EC119.011524
      Issue No: Vol. 294, No. 46 (2019)
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