scholarly journals GlnK facilitates the dynamic regulation of bacterial nitrogen assimilation

2017 ◽  
Author(s):  
Adam Gosztolai ◽  
Jörg Schumacher ◽  
Volker Behrends ◽  
Jacob G Bundy ◽  
Franziska Heydenreich ◽  
...  
Keyword(s):  
Nitrogen Assimilation ◽  
Ammonium Assimilation ◽  
Functional Link ◽  
Dynamic Regulation ◽  
Time Resolved ◽  
Allosteric Sites ◽  
Key Enzyme ◽  
In The Wild ◽  
Ammonium Level

AbstractAmmonium assimilation inE. coliis regulated by two paralogous proteins (GlnB and GlnK), which orchestrate interactions with regulators of gene expression, transport proteins and metabolic pathways. Yet how they conjointly modulate the activity of glutamine synthetase (GS), the key enzyme for nitrogen assimilation, is poorly understood. We combine experiments and theory to study the dynamic roles of GlnB and GlnK during nitrogen starvation and upshift. We measure time-resolvedin vivoconcentrations of metabolites, total and post-translationally modified proteins, and develop a concise biochemical model of GlnB and GlnK that incorporates competition for active and allosteric sites, as well as functional sequestration of GlnK. The model predicts the responses of GS, GlnB and GlnK under time-varying external ammonium level in the wild type and two genetic knock-outs. Our results show that GlnK is tightly regulated under nitrogen-rich conditions, yet it is expressed during ammonium run-out and starvation. This suggests a role for GlnK as a buffer of nitrogen shock after starvation, and provides a further functional link between nitrogen and carbon metabolisms.

Studies on possible routes of ammonium assimilation in soybean root nodule bacteroids

1973 ◽  
Vol 19 (12) ◽  
pp. 1493-1499 ◽  
Author(s):  
Stanley D. Dunn ◽  
Robert V. Klucas
Keyword(s):  
Glutamine Synthetase ◽  
Nitrogen Assimilation ◽  
Reductive Amination ◽  
Root Nodule ◽  
Root Nodules ◽  
Ammonium Assimilation ◽  
Kinetic Properties ◽  
Transferase Activity ◽  
Soybean Root

Glutamine amide–2-oxoglutarate aminotransferase NAD+ oxidoreductase (GOGAT), glutamine synthetase (GS), glutamate dehydrogenase (GD), and alanine dehydrogenase (AD) were studied in soybean root nodules. GS, GOGAT, and AD were present in bacteroids at levels that could account for ammonium assimilation, but GD activity was quite low. The total activities of GS and GD were higher in the cytosol than in the bacteroids by factors of 20 and 7, respectively, whereas GOGAT was not detected in the cytosol. GS (transferase activity) was inhibited by alanine, CTP, glycine, and tryptophan at 5 mM but was relatively unaffected by asparagine, aspartic acid, CMP, glucosamine, and histidine at 5 mM. GOGAT activity was unaffected by ATP, ADP, 8-hydroxyquinoline, and 1,10-phenanthroline but was inhibited by EDTA, citrate, and parachloromercuribenzoate. GOGAT activity (reductive amination) was also inhibited 97% by preincubation with 10−4 M azaserine for 30 min but GD activity was inhibited only 13%. The apparent Km values for NH4+ by AD was 7.4 × 10−3 M and by GD was 7.3 × 10−2 M while for glutamine by GOGAT it was 9.3 × 10−5 M. Activities and kinetic properties for these enzymes may suggest potential routes of nitrogen assimilation in vivo.

scholarly journals In Vivo Fluxes in the Ammonium-Assimilatory Pathways in Corynebacterium glutamicum Studied by15N Nuclear Magnetic Resonance

1999 ◽  
Vol 65 (3) ◽  
pp. 1099-1109 ◽  
Author(s):  
M. Tesch ◽  
A. A. de Graaf ◽  
H. Sahm
Keyword(s):  
Nuclear Magnetic Resonance ◽  
Magnetic Resonance ◽  
Corynebacterium Glutamicum ◽  
Ammonium Assimilation ◽  
Equation Model ◽  
Wild Type ◽  
In The Wild ◽  
High Fraction ◽  
Nuclear Magnetic

ABSTRACT Glutamate dehydrogenase (GDH) and glutamine synthetase (GS)–glutamine 2-oxoglutarate-aminotransferase (GOGAT) represent the two main pathways of ammonium assimilation in Corynebacterium glutamicum. In this study, the ammonium assimilating fluxes in vivo in the wild-type ATCC 13032 strain and its GDH mutant were quantitated in continuous cultures. To do this, the incorporation of15N label from [15N]ammonium in glutamate and glutamine was monitored with a time resolution of about 10 min with in vivo 15N nuclear magnetic resonance (NMR) used in combination with a recently developed high-cell-density membrane-cyclone NMR bioreactor system. The data were used to tune a standard differential equation model of ammonium assimilation that comprised ammonia transmembrane diffusion, GDH, GS, GOGAT, and glutamine amidotransferases, as well as the anabolic incorporation of glutamate and glutamine into biomass. The results provided a detailed picture of the fluxes involved in ammonium assimilation in the two different C. glutamicumstrains in vivo. In both strains, transmembrane equilibration of 100 mM [15N]ammonium took less than 2 min. In the wild type, an unexpectedly high fraction of 28% of the NH4 + was assimilated via the GS reaction in glutamine, while 72% were assimilated by the reversible GDH reaction via glutamate. GOGAT was inactive. The analysis identified glutamine as an important nitrogen donor in amidotransferase reactions. The experimentally determined amount of 28% of nitrogen assimilated via glutamine is close to a theoretical 21% calculated from the high peptidoglycan content of C. glutamicum. In the GDH mutant, glutamate was exclusively synthesized over the GS/GOGAT pathway. Its level was threefold reduced compared to the wild type.

scholarly journals IPMK physically binds to the SWI/SNF complex and modulates BRG1 occupancy

2021 ◽  
Author(s):  
Jiyoon Beon ◽  
Sungwook Han ◽  
Seung Eun Park ◽  
Kwangbeom Hyun ◽  
Song-Yi Lee ◽  
...  
Keyword(s):  
Transcriptional Control ◽  
Critical Role ◽  
Embryonic Stem ◽  
Chromatin Accessibility ◽  
Inositol Polyphosphate ◽  
Functional Link ◽  
A Genome ◽  
Key Enzyme

ABSTRACTInositol polyphosphate multikinase (IPMK), a key enzyme in the inositol polyphosphate (IP) metabolism, is a pleiotropic signaling factor involved in major biological events including transcriptional control. In yeasts, IPMK and its IP products were known to promote the activity of SWI/SNF chromatin remodeling complex, which plays a critical role in gene expression by regulating chromatin accessibility. However, the direct linkage between IPMK and chromatin remodelers remains unclear, raising a question on how IPMK contributes to the transcriptional regulation in mammals. By employing unbiased screenings and in vivo/in vitro immunoprecipitations, here we demonstrated that IPMK physically associates with native mammalian SWI/SNF complexes by directly binding to SMARCB1, BRG1, and SMARCC1. Furthermore, we identified the specific domains required for the IPMK-SMARCB1 binding. Notably, using CUT&RUN and ATAC-seq assays, we discovered that IPMK co-localizes with BRG1 and regulates BRG1 localization as well as BRG1-mediated chromatin accessibility in a genome-wide manner (including promoter-TSS) in mouse embryonic stem cells. Finally, our mRNA-seq analyses revealed that IPMK and SMARCB1 regulate common gene sets, validating a functional link between IPMK and SWI/SNF complex. Together, these findings establish an importance of IPMK in promoter targeting of the SWI/SNF complex, thereby contributing to SWI/SNF-meditated chromatin accessibility and transcription.

Approach in improving potency and selectivity of kinase inhibitors: Allosteric kinase inhibitors

2020 ◽  
Vol 21 ◽  
Author(s):  
Shangfei Wei ◽  
Tianming Zhao ◽  
Jie Wang ◽  
Xin Zhai
Keyword(s):  
Protein Interactions ◽  
Kinase Inhibitors ◽  
Binding Modes ◽  
Allosteric Inhibitors ◽  
Wide Range ◽  
Allosteric Sites ◽  
Protein Functions ◽  
Regulate Protein

: Allostery is an efficient and particular regulatory mechanism to regulate protein functions. Different from conserved orthosteric sites, allosteric sites have distinctive functional mechanism to form the complex regulatory network. In drug discovery, kinase inhibitors targeting the allosteric pockets have received extensive attention for the advantages of high selectivity and low toxicity. The approval of trametinib as the first allosteric inhibitor validated that allosteric inhibitors could be used as effective therapeutic drugs for treatment of diseases. To date, a wide range of allosteric inhibitors have been identified. In this perspective, we outline different binding modes and potential advantages of allosteric inhibitors. In the meantime, the research processes of typical and novel allosteric inhibitors are described briefly in terms of structureactivity relationships, ligand-protein interactions and in vitro and in vivo activity. Additionally, challenges as well as opportunities are presented.

scholarly journals Real-time observation of tetrapyrrole binding to an engineered bacterial phytochrome

2021 ◽  
Vol 4 (1) ◽  
Author(s):  
Yusaku Hontani ◽  
Mikhail Baloban ◽  
Francisco Velazquez Escobar ◽  
Swetta A. Jansen ◽  
Daria M. Shcherbakova ◽  
...  
Keyword(s):  
Real Time ◽  
Rational Design ◽  
Near Infrared ◽  
Fluorescent Proteins ◽  
Covalent Bond ◽  
Binding Pocket ◽  
Tissue Imaging ◽  
Covalent Attachment ◽  
Time Resolved

AbstractNear-infrared fluorescent proteins (NIR FPs) engineered from bacterial phytochromes are widely used for structural and functional deep-tissue imaging in vivo. To fluoresce, NIR FPs covalently bind a chromophore, such as biliverdin IXa tetrapyrrole. The efficiency of biliverdin binding directly affects the fluorescence properties, rendering understanding of its molecular mechanism of major importance. miRFP proteins constitute a family of bright monomeric NIR FPs that comprise a Per-ARNT-Sim (PAS) and cGMP-specific phosphodiesterases - Adenylyl cyclases - FhlA (GAF) domain. Here, we structurally analyze biliverdin binding to miRFPs in real time using time-resolved stimulated Raman spectroscopy and quantum mechanics/molecular mechanics (QM/MM) calculations. Biliverdin undergoes isomerization, localization to its binding pocket, and pyrrolenine nitrogen protonation in <1 min, followed by hydrogen bond rearrangement in ~2 min. The covalent attachment to a cysteine in the GAF domain was detected in 4.3 min and 19 min in miRFP670 and its C20A mutant, respectively. In miRFP670, a second C–S covalent bond formation to a cysteine in the PAS domain occurred in 14 min, providing a rigid tetrapyrrole structure with high brightness. Our findings provide insights for the rational design of NIR FPs and a novel method to assess cofactor binding to light-sensitive proteins.

scholarly journals Influence of Oxidative Stress on Time-Resolved Oxygen Detection by [Ru(Phen)3]2+ In Vivo and In Vitro

Molecules ◽  
2021 ◽  
Vol 26 (2) ◽  
pp. 485
Author(s):  
Veronika Huntosova ◽  
Denis Horvath ◽  
Robert Seliga ◽  
Georges Wagnieres
Keyword(s):  
Oxidative Stress ◽  
Tissue Oxygenation ◽  
Intact Cell ◽  
Molecular Probes ◽  
Stress Factors ◽  
Time Resolved ◽  
Invasive Approach ◽  
Oxygen Detection

Detection of tissue and cell oxygenation is of high importance in fundamental biological and in many medical applications, particularly for monitoring dysfunction in the early stages of cancer. Measurements of the luminescence lifetimes of molecular probes offer a very promising and non-invasive approach to estimate tissue and cell oxygenation in vivo and in vitro. We optimized the evaluation of oxygen detection in vivo by [Ru(Phen)3]2+ in the chicken embryo chorioallantoic membrane model. Its luminescence lifetimes measured in the CAM were analyzed through hierarchical clustering. The detection of the tissue oxygenation at the oxidative stress conditions is still challenging. We applied simultaneous time-resolved recording of the mitochondrial probe MitoTrackerTM OrangeCMTMRos fluorescence and [Ru(Phen)3]2+ phosphorescence imaging in the intact cell without affecting the sensitivities of these molecular probes. [Ru(Phen)3]2+ was demonstrated to be suitable for in vitro detection of oxygen under various stress factors that mimic oxidative stress: other molecular sensors, H2O2, and curcumin-mediated photodynamic therapy in glioma cancer cells. Low phototoxicities of the molecular probes were finally observed. Our study offers a high potential for the application and generalization of tissue oxygenation as an innovative approach based on the similarities between interdependent biological influences. It is particularly suitable for therapeutic approaches targeting metabolic alterations as well as oxygen, glucose, or lipid deprivation.

scholarly journals USP29-mediated HIF1α stabilization is associated with Sorafenib resistance of hepatocellular carcinoma cells by upregulating glycolysis

Oncogenesis ◽  
2021 ◽  
Vol 10 (7) ◽  
Author(s):  
Ruize Gao ◽  
David Buechel ◽  
Ravi K. R. Kalathur ◽  
Marco F. Morini ◽  
Mairene Coto-Llerena ◽  
...  
Keyword(s):  
Hepatocellular Carcinoma ◽  
Transcriptional Activity ◽  
Kinase Inhibitor ◽  
Aerobic Glycolysis ◽  
Hypoxia Inducible Factor ◽  
Aberrant Expression ◽  
Key Enzyme ◽  
Hcc Cells

AbstractUnderstanding the mechanisms underlying evasive resistance in cancer is an unmet medical need to improve the efficacy of current therapies. In hepatocellular carcinoma (HCC), aberrant expression of hypoxia-inducible factor 1 α (HIF1α) and increased aerobic glycolysis metabolism are drivers of resistance to therapy with the multi-kinase inhibitor Sorafenib. However, it has remained unknown how HIF1α is activated and how its activity and the subsequent induction of aerobic glycolysis promote Sorafenib resistance in HCC. Here, we report the ubiquitin-specific peptidase USP29 as a new regulator of HIF1α and of aerobic glycolysis during the development of Sorafenib resistance in HCC. In particular, we identified USP29 as a critical deubiquitylase (DUB) of HIF1α, which directly deubiquitylates and stabilizes HIF1α and, thus, promotes its transcriptional activity. Among the transcriptional targets of HIF1α is the gene encoding hexokinase 2 (HK2), a key enzyme of the glycolytic pathway. The absence of USP29, and thus of HIF1α transcriptional activity, reduces the levels of aerobic glycolysis and restores sensitivity to Sorafenib in Sorafenib-resistant HCC cells in vitro and in xenograft transplantation mouse models in vivo. Notably, the absence of USP29 and high HK2 expression levels correlate with the response of HCC patients to Sorafenib therapy. Together, the data demonstrate that, as a DUB of HIF1α, USP29 promotes Sorafenib resistance in HCC cells, in parts by upregulating glycolysis, thereby opening new avenues for therapeutically targeting Sorafenib-resistant HCC in patients.

scholarly journals A Versatile Setup for Time-Resolved Functional Near Infrared Spectroscopy Based on Fast-Gated Single-Photon Avalanche Diode and on Four-Wave Mixing Laser

2019 ◽  
Vol 9 (11) ◽  
pp. 2366 ◽  
Author(s):  
Laura Di Sieno ◽  
Alberto Dalla Mora ◽  
Alessandro Torricelli ◽  
Lorenzo Spinelli ◽  
Rebecca Re ◽  
...  
Keyword(s):  
Infrared Spectroscopy ◽  
Near Infrared Spectroscopy ◽  
Near Infrared ◽  
Single Photon ◽  
Wave Mixing ◽  
Functional Near Infrared Spectroscopy ◽  
Time Resolved ◽  
Diffusive Medium ◽  
Spectroscopy System

In this paper, a time-domain fast gated near-infrared spectroscopy system is presented. The system is composed of a fiber-based laser providing two pulsed sources and two fast gated detectors. The system is characterized on phantoms and was tested in vivo, showing how the gating approach can improve the contrast and contrast-to-noise-ratio for detection of absorption perturbation inside a diffusive medium, regardless of source-detector separation.

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