scholarly journals Activation of GCN2 by the ribosomal P-stalk

2019 ◽  
Vol 116 (11) ◽  
pp. 4946-4954 ◽  
Author(s):  
Alison J. Inglis ◽  
Glenn R. Masson ◽  
Sichen Shao ◽  
Olga Perisic ◽  
Stephen H. McLaughlin ◽  
...  
Keyword(s):  
Conformational Changes ◽  
Principal Component ◽  
Protein Translation ◽  
Translation Initiation Factor ◽  
Initiation Factor ◽  
Exchange Mass Spectrometry ◽  
Hydrogen Deuterium ◽  
Hdx Ms ◽  
Ribosomal P

Cells dynamically adjust their protein translation profile to maintain homeostasis in changing environments. During nutrient stress, the kinase general control nonderepressible 2 (GCN2) phosphorylates translation initiation factor eIF2α, initiating the integrated stress response (ISR). To examine the mechanism of GCN2 activation, we have reconstituted this process in vitro, using purified components. We find that recombinant human GCN2 is potently stimulated by ribosomes and, to a lesser extent, by tRNA. Hydrogen/deuterium exchange–mass spectrometry (HDX-MS) mapped GCN2–ribosome interactions to domain II of the uL10 subunit of the ribosomal P-stalk. Using recombinant, purified P-stalk, we showed that this domain of uL10 is the principal component of binding to GCN2; however, the conserved 14-residue C-terminal tails (CTTs) in the P1 and P2 P-stalk proteins are also essential for GCN2 activation. The HisRS-like and kinase domains of GCN2 show conformational changes upon binding recombinant P-stalk complex. Given that the ribosomal P-stalk stimulates the GTPase activity of elongation factors during translation, we propose that the P-stalk could link GCN2 activation to translational stress, leading to initiation of ISR.

scholarly journals Modulation of PTH1R signaling by an ECD binding antibody results in inhibition of β-arrestin 2 coupling

2019 ◽  
Vol 9 (1) ◽  
Author(s):  
Kaushik Sarkar ◽  
Lisa Joedicke ◽  
Marta Westwood ◽  
Rebecca Burnley ◽  
Michael Wright ◽  
...  
Keyword(s):  
Parathyroid Hormone ◽  
Conformational Changes ◽  
Transmembrane Domain ◽  
Receptor Activation ◽  
Parathyroid Hormone Receptor ◽  
Exchange Mass Spectrometry ◽  
Gpcr Activation ◽  
Hydrogen Deuterium ◽  
Binding Antibody ◽  
Hdx Ms

Abstract Parathyroid hormone receptor 1 (PTH1R) belongs to the secretin class of G protein coupled receptors (GPCRs) and natively binds parathyroid hormone (PTH) and parathyroid hormone related peptide (PTHrP). Ligand binding to PTH1R involves binding to the large extracellular domain (ECD) and the orthosteric pocket, inducing conformational changes in the transmembrane domain and receptor activation. PTH1R regulates bone metabolism, signaling mainly through Gs and Gq/11 G-proteins. Here, we used phage display to generate PTH1R ECD-specific antibodies with the aim of modulating receptor functionality. We identified ECD-scFvhFc, which exhibited high affinity binding to both the isolated ECD and to the full-length receptor in styrene-maleic acid (SMA) lipid particles. Epitope mapping using hydrogen-deuterium exchange mass spectrometry (HDX-MS) indicates that the α1 helix of the ECD is ECD-scFvhFc’s epitope which may partially overlap with the known PTH (1–34) binding site. However, PTH (1–34)-mediated Gs activation is Undisturbed by ECD-scFvhFc binding. In contrast, ECD-scFvhFc potently inhibits β-arrestin-2 recruitment after PTH (1–34)-driven receptor activation and thus represents the first monoclonal antibody to selectively inhibit distinct PTH1R signaling pathways. Given the complexity of PTH1R signaling and the emerging importance of biased GPCR activation in drug development, ECD-scFvhFc could be a valuable tool to study PTH1R signaling bias.

scholarly journals Activation of phospholipase C β by Gβγ and Gαq involves C-terminal rearrangement to release auto-inhibition

2019 ◽  
Author(s):  
Isaac Fisher ◽  
Meredith Jenkins ◽  
Greg Tall ◽  
John E Burke ◽  
Alan V. Smrcka
Keyword(s):  
Phospholipase C ◽  
Inositol Phosphates ◽  
G Protein Coupled Receptors ◽  
Exchange Mass Spectrometry ◽  
Hydrogen Deuterium ◽  
Deuterium Exchange Mass Spectrometry ◽  
Hdx Ms ◽  
G Protein Coupled ◽  
Inhibitory Interactions

AbstractPhospholipase C (PLC) enzymes hydrolyse phosphoinositide lipids to inositol phosphates and diacylglycerol. Direct activation of PLCβ by Gαq and/or Gβγ subunits mediates signalling by Gq and some Gi coupled G protein-coupled receptors (GPCRs), respectively. PLCβ isoforms contain a unique C-terminal extension, consisting of proximal and distal C-terminal domains (CTD) separated by a flexible linker. The structure of PLCβ3 bound to Gαq is known, however, for both Gαq and Gβγ, the mechanism for PLCβ activation on membranes is unknown. We examined PLCβ2 dynamics on membranes using hydrogen deuterium exchange mass spectrometry (HDX-MS). Gβγ caused a robust increase in dynamics of the distal C-terminal domain (CTD). Gαq showed decreased deuterium incorporation at the Gαq binding site on PLCβ. In vitro Gβγ-dependent activation of PLC is inhibited by the distal CTD. The results suggest that disruption of auto-inhibitory interactions with the CTD, respectively, leads to increased PLCβ hydrolase activity.

scholarly journals The Impact of Immunoglobulin G1 Fc Sialylation on Backbone Amide H/D Exchange

Antibodies ◽  
2019 ◽  
Vol 8 (4) ◽  
pp. 49
Author(s):  
Kuhne ◽  
Bonnington ◽  
Malik ◽  
Thomann ◽  
Avenal ◽  
...  
Keyword(s):  
Conformational Changes ◽  
Structural Information ◽  
Binding Properties ◽  
Engineering Technology ◽  
Exchange Mass Spectrometry ◽  
Structural Impact ◽  
Hydrogen Deuterium ◽  
Deuterium Exchange Mass Spectrometry ◽  
The Impact

The usefulness of higher-order structural information provided by hydrogen/deuterium exchange-mass spectrometry (H/DX-MS) for the structural impact analyses of chemical and post-translational antibody modifications has been demonstrated in various studies. However, the structure–function assessment for protein drugs in biopharmaceutical research and development is often impeded by the relatively low-abundance (below 5%) of critical quality attributes or by overlapping effects of modifications, such as glycosylation, with chemical amino acid modifications; e.g., oxidation or deamidation. We present results demonstrating the applicability of the H/DX-MS technique to monitor conformational changes of specific Fc glycosylation variants produced by in vitro glyco-engineering technology. A trend towards less H/DX in Fc Cγ2 domain segments correlating with larger glycan structures could be confirmed. Furthermore, significant deuterium uptake differences and corresponding binding properties to Fc receptors (as monitored by SPR) between α-2,3- and α-2,6-sialylated Fc glycosylation variants were verified at sensitive levels.

Protein 4.1R binding to eIF3-p44 suggests an interaction between the cytoskeletal network and the translation apparatus

Blood ◽  
2000 ◽  
Vol 96 (2) ◽  
pp. 747-753 ◽  
Author(s):  
Chia-Lung Hou ◽  
Chieh-ju C. Tang ◽  
Steve R. Roffler ◽  
Tang K. Tang
Keyword(s):  
Translation Initiation ◽  
Protein Translation ◽  
Translation Initiation Factor ◽  
Initiation Factor ◽  
Translation System ◽  
Cdna Clones ◽  
Eukaryotic Translation ◽  
Translation Apparatus ◽  
Direct Association

Erythroid protein 4.1 (4.1R) is an 80-kd cytoskeletal protein that stabilizes the membrane-skeletal network structure underlying the lipid bilayer. Using the carboxyl terminal domain (22/24 kd) of 4.1R as bait in a yeast 2-hybrid screen, we isolated cDNA clones encoding a polypeptide of eIF3-p44, which represents a subunit of a eukaryotic translation initiation factor 3 (eIF3) complex. The eIF3 complex consists of at least 10 subunits that play an essential role in the pathway of protein translation initiation. Northern blot analysis revealed that eIF3-p44 (approximately 1.35 kb) is constitutively expressed in many tissues. The essential sequence for this interaction was mapped to the carboxyl-terminus of 4.1R (residues 525-622) and a region (residues 54-321) of eIF3-p44. The direct association between 4.1R and eIF3-p44 was further confirmed by in vitro binding assays and coimmunoprecipitation studies. To characterize the functions of eIF3-p44, we depleted eIF3-p44 from rabbit reticulocyte lysates either by anti-eIF3-p44 antibody or by GST/4.1R-80 fusion protein. Our results show that the eIF3-p44 depleted cell-free translation system was unable to synthesize proteins efficiently. The direct association between 4.1R and elF3-p44 suggests that 4.1R may act as an anchor protein that links the cytoskeleton network to the translation apparatus.

scholarly journals Crl activates transcription by stabilizing the active conformation of the master stress factor σS

2019 ◽  
Author(s):  
Juncao Xu ◽  
Kaijie Cui ◽  
Liqiang Shen ◽  
Jing Shi ◽  
Lingting Li ◽  
...  
Keyword(s):  
Transcription Initiation ◽  
Transcription Activation ◽  
Initiation Factor ◽  
Bacterial Cells ◽  
E Coli ◽  
Exchange Mass Spectrometry ◽  
Transcription Initiation Factor ◽  
Hydrogen Deuterium ◽  
Promoter Dna ◽  
Hdx Ms

SUMMARYσS is a master transcription initiation factor that protects bacterial cells from various harmful environmental stresses and antibiotic pressure. Although its mechanism remains unclear, it is known that full activation of σS-mediated transcription requires a σS-specific activator, Crl. In this study, we determined a 3.80 Å cryo-EM structure of an E. coli transcription activation complex (E. coli Crl-TAC) comprising E. coli σS-RNAP holoenzyme, Crl, and a nucleic-acid scaffold. The structure reveals that Crl interacts with the domain 2 of σS (σS2), sharing no interaction with promoter DNA. Subsequent hydrogen-deuterium exchange mass spectrometry (HDX-MS) results indicate that Crl stabilizes key structural motifs of σS2 to promote the assembly of σS-RNAP holoenzyme and also to facilitate formation of the RNA polymerase-promoter DNA open complex (RPo). Our study demonstrates a unique DNA contact-independent mechanism of transcription activation, thereby defining a previously unrecognized mode of transcription activation in cells.

scholarly journals HDX-MS reveals nucleotide-dependent, anti-correlated opening and closure of SecA and SecY channels of the bacterial translocon

eLife ◽  
2019 ◽  
Vol 8 ◽  
Author(s):  
Zainab Ahdash ◽  
Euan Pyle ◽  
William John Allen ◽  
Robin A Corey ◽  
Ian Collinson ◽  
...  
Keyword(s):  
Conformational Changes ◽  
Opposite Effect ◽  
Protein Transport ◽  
Protein Translocation ◽  
Brownian Ratchet ◽  
Exchange Mass Spectrometry ◽  
Hydrogen Deuterium ◽  
Deuterium Exchange Mass Spectrometry ◽  
Dependent Transport ◽  
Hdx Ms

The bacterial Sec translocon is a multi-protein complex responsible for translocating diverse proteins across the plasma membrane. For post-translational protein translocation, the Sec-channel – SecYEG – associates with the motor protein SecA to mediate the ATP-dependent transport of pre-proteins across the membrane. Previously, a diffusional-based Brownian ratchet mechanism for protein secretion has been proposed; the structural dynamics required to facilitate this mechanism remain unknown. Here, we employ hydrogen-deuterium exchange mass spectrometry (HDX-MS) to reveal striking nucleotide-dependent conformational changes in the Sec protein-channel from Escherichia coli. In addition to the ATP-dependent opening of SecY, reported previously, we observe a counteracting, and ATP-dependent, constriction of SecA around the pre-protein. ATP binding causes SecY to open and SecA to close; while, ADP produced by hydrolysis, has the opposite effect. This alternating behaviour could help impose the directionality of the Brownian ratchet for protein transport through the Sec machinery.

Fluorescence Studies on Association of Human Translation Initiation Factor eIF4E with mRNA cap-Analogues

1999 ◽  
Vol 54 (3-4) ◽  
pp. 278-284 ◽  
Author(s):  
Zbigniew Wieczorek ◽  
Anna Niedźwiecka-Kornaś ◽  
Lidia Chlebicka ◽  
Marzena Jankowska ◽  
Katarzyna Kiraga ◽  
...  
Keyword(s):  
Translation Initiation ◽  
Biological Activities ◽  
Methodological Approach ◽  
Protein Translation ◽  
Structural Features ◽  
Translation Initiation Factor ◽  
Initiation Factor ◽  
Protein Fluorescence ◽  
Fluorescence Studies

Binding of a long series of mono- and dinucleotide analogues of the 7-methylguanosine containing 5′-mRNA-cap to human protein translation initiation factor eIF4E has been investigated by means of fluorescence. A new methodological approach in gathering and analysis of the fluorescence data provided us with very accurate values of the association equilibrium constant K and normalized, maximal quenching of the protein fluorescence ΔFmax, during titration of eIF4E by various cap-analogues. The results confirm participation of at least two conserved tryptophan residues of eIF4E in interaction with 7-methylguanine, as has been described recently for murine eIF4E, complexed with 7-methyl-GDP in crystal (Marcotrigiano et al., 1997, Cell 89, 951), and for yeast eIF4E, complexed with the same ligand in solution (Matsuo et al., 1997, Nature Struct. Biol. 4, 717). On the other hand binding by eIF4E of unmethylated guanine nucleotides and N2,N2,7-trimethylguanine containing nucleotides differ substantially from the way of binding of the regular mRNA-cap. Influence of the structural features of the cap-analogues, especially the type of the second nucleoside in the dinucleotide caps, on their association with eIF4E and biological activities in in vitro protein translation systems has been discussed in light of the known structures of the eIF4E -7- methyl-GDP complexes in crystal and solution.

scholarly journals Hydrogen-deuterium exchange mass spectrometry captures distinct dynamics upon substrate and inhibitor binding to a transporter

2020 ◽  
Vol 11 (1) ◽  
Author(s):  
Ruyu Jia ◽  
Chloe Martens ◽  
Mrinal Shekhar ◽  
Shashank Pant ◽  
Grant A. Pellowe ◽  
...  
Keyword(s):  
Conformational Changes ◽  
Conformational Transition ◽  
Substrate Binding ◽  
Conformational Dynamics ◽  
Md Simulations ◽  
Allosteric Coupling ◽  
Exchange Mass Spectrometry ◽  
Hydrogen Deuterium ◽  
Deuterium Exchange Mass Spectrometry ◽  
Hdx Ms

AbstractProton-coupled transporters use transmembrane proton gradients to power active transport of nutrients inside the cell. High-resolution structures often fail to capture the coupling between proton and ligand binding, and conformational changes associated with transport. We combine HDX-MS with mutagenesis and MD simulations to dissect the molecular mechanism of the prototypical transporter XylE. We show that protonation of a conserved aspartate triggers conformational transition from outward-facing to inward-facing state. This transition only occurs in the presence of substrate xylose, while the inhibitor glucose locks the transporter in the outward-facing state. MD simulations corroborate the experiments by showing that only the combination of protonation and xylose binding, and not glucose, sets up the transporter for conformational switch. Overall, we demonstrate the unique ability of HDX-MS to distinguish between the conformational dynamics of inhibitor and substrate binding, and show that a specific allosteric coupling between substrate binding and protonation is a key step to initiate transport.

scholarly journals Discovery of a small molecule that inhibits bacterial ribosome biogenesis

eLife ◽  
2014 ◽  
Vol 3 ◽  
Author(s):  
Jonathan M Stokes ◽  
Joseph H Davis ◽  
Chand S Mangat ◽  
James R Williamson ◽  
Eric D Brown
Keyword(s):  
Small Molecule ◽  
Ribosome Biogenesis ◽  
Anticonvulsant Drug ◽  
Protein Translation ◽  
Translation Initiation Factor ◽  
Initiation Factor ◽  
Bacterial Ribosome ◽  
Cold Sensitive

While small molecule inhibitors of the bacterial ribosome have been instrumental in understanding protein translation, no such probes exist to study ribosome biogenesis. We screened a diverse chemical collection that included previously approved drugs for compounds that induced cold sensitive growth inhibition in the model bacterium Escherichia coli. Among the most cold sensitive was lamotrigine, an anticonvulsant drug. Lamotrigine treatment resulted in the rapid accumulation of immature 30S and 50S ribosomal subunits at 15°C. Importantly, this was not the result of translation inhibition, as lamotrigine was incapable of perturbing protein synthesis in vivo or in vitro. Spontaneous suppressor mutations blocking lamotrigine activity mapped solely to the poorly characterized domain II of translation initiation factor IF2 and prevented the binding of lamotrigine to IF2 in vitro. This work establishes lamotrigine as a widely available chemical probe of bacterial ribosome biogenesis and suggests a role for E. coli IF2 in ribosome assembly.

scholarly journals The circular RNA circ-ERBIN promotes growth and metastasis of colorectal cancer by miR-125a-5p and miR-138-5p/4EBP-1 mediated cap-independent HIF-1α translation

2020 ◽  
Vol 19 (1) ◽  
Author(s):  
Liang-Yan Chen ◽  
Lian Wang ◽  
Yue-Xiang Ren ◽  
Zheng Pang ◽  
Yao Liu ◽  
...  
Keyword(s):  
Colorectal Cancer ◽  
Protein Translation ◽  
Circular Rna ◽  
Translation Initiation Factor ◽  
Initiation Factor ◽  
Luciferase Reporter ◽  
Western Blots ◽  
Eukaryotic Translation

Abstract Background Circular RNA (circRNAs) and hypoxia have been found to play the key roles in the pathogenesis and progression of cancer including colorectal cancer (CRC). However, the expressions and functions of the specific circRNAs in regulating hypoxia-involved CRC metastasis, and the circRNAs that are relevant to regulate HIF-1α levels in CRC remain elusive. Methods qRT-PCR was used to detect the expression of circRNAs and mRNA in CRC cells and tissues. Fluorescence in situ hybridization (FISH) was used to analyze the location of circ-ERBIN. Function-based experiments were performed using circ-ERBIN overexpression and knockdown cell lines in vitro and in vivo, including CCK8, colony formation, EdU assay, transwell, tumor growth and metastasis models. Mechanistically, luciferase reporter assay, western blots and immunohistochemical stainings were performed. Results Circ-Erbin was highly expressed in the CRC cells and Circ-Erbin overexpression facilitated the proliferation, migration and metastasis of CRC in vitro and in vivo. Notably, circ-Erbin overexpression significantly promoted angiogenesis by increasing the expression of hypoxia induced factor (HIF-1α) in CRC. Mechanistically, circ-Erbin accelerated a cap-independent protein translation of HIF-1α in CRC cells as the sponges of miR-125a-5p and miR-138-5p, which synergistically targeted eukaryotic translation initiation factor 4E binding protein 1(4EBP-1). Conclusions Our findings uncover a key mechanism for circ-Erbin mediated HIF-1α activation by miR-125a-5p-5p/miR-138-5p/4EBP-1 axis and circ-ERBIN is a potential target for CRC treatment.

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