Summary of Study ST001375

This data is available at the NIH Common Fund's National Metabolomics Data Repository (NMDR) website, the Metabolomics Workbench,, where it has been assigned Project ID PR000941. The data can be accessed directly via it's Project DOI: 10.21228/M8296W This work is supported by NIH grant, U2C- DK119886.


Perform statistical analysis  |  Show all samples  |  Show named metabolites  |  Download named metabolite data  
Download mwTab file (text)   |  Download mwTab file(JSON)   |  Download data files (Contains raw data)
Study IDST001375
Study TitleFructosamine-3-kinase (FN3K) KO in HepG2 liver cancer cells
Study SummaryFructosamine-3-kinases (FN3Ks) are a family of metabolic kinases which are evolutionarily related to eukaryotic protein kinases. Aberrant regulation of these kinases by altered redox homeostasis is a major contributing factor in aging and disease. However, the mechanisms of regulation and cellular functions of these kinases are not known. Bioinformatic analyses of cancer cell lines identified significant overexpression of FN3K in liver and eye cancer cells. To assess the functional significance of this increased expression, a CRISPR knockout of FN3K (FN3K-KO) was generated in the HepG2 liver cancer cell line. The metabolome was compared between FN3K-KO and WT HepG2 cells using untargeted 1H NMR metabolomics. This revealed significant differences in several metabolites that suggest a role for FN3K in regulating redox and energy balance in HepG2 cells.
University of Georgia
DepartmentComplex Carbohydrate Research Center
Last NameColonna
First NameMaxwell
Address315 Riverbend Rd, Athens, GA 30602
Submit Date2020-05-08
Num Groups2
PublicationsA redox-active switch in Fructosamine-3-kinases expands the regulatory repertoire of the protein kinase super-family
Raw Data AvailableYes
Raw Data File Type(s)fid
Analysis Type DetailNMR
Release Date2020-07-07
Release Version1
Maxwell Colonna Maxwell Colonna application/zip

Select appropriate tab below to view additional metadata details:


Project ID:PR000941
Project DOI:doi: 10.21228/M8296W
Project Title:A redox-active switch in Fructosamine-3-kinases expands the regulatory repertoire of the protein kinase super-family
Project Type:Structural Biology, enzyme characterization
Project Summary:Aberrant regulation of metabolic kinases by altered redox homeostasis is a major contributing factor in aging and disease such as diabetes. However, the biochemical mechanisms by which metabolic kinases are regulated under oxidative stress is poorly understood. In this study, we demonstrate that the catalytic activity of a conserved family of Fructosamine-3-kinases (FN3Ks), which are evolutionarily related to eukaryotic protein kinases (ePKs), are regulated by redox-active cysteines in the kinase domain. By solving the crystal structure of FN3K homolog from Arabidopsis thaliana (AtFN3K), we demonstrate that it forms an unexpected strand-exchange dimer in which the ATP binding P-loop and adjoining beta strands are swapped between two chains in the dimer. This dimeric configuration is characterized by strained inter-chain disulfide bonds that stabilize the P-loop in an extended conformation. Mutational analysis and solution studies confirm that the strained disulfides function as redox “switches” to reversibly regulate FN3K activity and dimerization. Consistently, we find that human FN3K, which contains an equivalent P-loop Cys, is also redox-sensitive, whereas ancestral bacterial FN3K homologs, which lack a P-loop Cys, are not. Furthermore, CRISPR knockout of FN3K in human HepG2 cells results in significant upregulation of redox metabolites including glutathione. We propose that redox regulation evolved progressively in FN3Ks in response to changing cellular redox conditions. Our studies provide important new insights into the origin and evolution of redox regulation in the protein kinase superfamily and open new avenues for targeting human FN3K in diabetic complications.
Institute:University of Georgia
Department:Biochemistry and Molecular Biology
Last Name:Kannan
First Name:Natarajan
Address:B122 Life Sciences Bldg. University of Georgia Athens, GA 30602
Funding Source:MCB-1149106; R01GM114409
Publications:A redox-active switch in Fructosamine-3-kinases expands the regulatory repertoire of the protein kinase super-family
Contributors:Safal Shrestha, Samiksha Katiyar, Carlos E. Sanz-Rodriquez, Nolan R. Kemppinen, Hyun W. Kim, Renuka Kadirvelraj, Charalampos Panagos, Neda Keyhaninejad, Maxwell Colonna, Pradeep Chopra, Dominic P. Byrne, Geert J. Boons, Esther V. Knaap, Patrick A. Eyers, Arthur S. Edison, Zachary A. Wood


Subject ID:SU001449
Subject Type:Cultured cells
Subject Species:Homo sapiens
Taxonomy ID:9606
Genotype Strain:HepG2


Subject type: Cultured cells; Subject species: Homo sapiens (Factor headings shown in green)

mb_sample_id local_sample_id Genotype
SA100481FN3K_9FN3K KO
SA100482FN3K_10FN3K KO
SA100483FN3K_1FN3K KO
SA100484FN3K_7FN3K KO
SA100485FN3K_8FN3K KO
SA100486FN3K_6FN3K KO
SA100487FN3K_2FN3K KO
SA100488FN3K_4FN3K KO
SA100489FN3K_3FN3K KO
SA100490FN3K_5FN3K KO
Showing results 1 to 20 of 20


Collection ID:CO001444
Collection Summary:Cell cultures for both cell lines were grown simultaneously with identical media components. Upon achieving ~80% confluence in 10cm culture dish, culture media was removed and cell monolayer was washed with cold phosphate buffered saline. Cells were then quenched by addition ice-cold 80% methanol extraction solvent, scraped from the culture dish, and flash-frozen in liquid nitrogen.
Sample Type:HepG2 cells
Storage Conditions:-80℃


Treatment ID:TR001464
Treatment Summary:None

Sample Preparation:

Sampleprep ID:SP001457
Sampleprep Summary:Aqueous metabolites were extracted by vortexing cell pellets in the extraction solvent, pelleting cell debris and collecting the supernatant. Approximately 10% of the supernatant volume was taken from each sample to form an internal pooled sample. The solvent was then evaporated to produce dried extracts.
Sampleprep Protocol Filename:cell_protocol.pdf
Processing Storage Conditions:On ice
Extraction Method:Vortex
Extract Enrichment:Polar
Extract Storage:-80℃
Sample Resuspension:Deuterium oxide phosphate buffer


Analysis ID:AN002295
Laboratory Name:Edison
Analysis Type:NMR
Acquisition Date:22-Feb-18
Software Version:Topspin 4.0.1
Operator Name:Maxwell Colonna
Data Format:Bruker
Num Factors:2
Num Metabolites:24


NMR ID:NM000164
Analysis ID:AN002295
Instrument Name:Bruker Avance
Instrument Type:FT-NMR
NMR Experiment Type:1D-1H
Field Frequency Lock:Deuterium
Spectrometer Frequency:800 MHz
NMR Probe:3mm Cryoprobe
NMR Solvent:D2O Phosphate buffer
NMR Tube Size:3mm SampleJet
Shimming Method:Topshim
Pulse Sequence:noesypr1d
Water Suppression:Presaturation
Pulse Width:8.22 us
Power Level:11.117 W
Offset Frequency:3762.64
Presaturation Power Level:3.00E-05
Chemical Shift Ref Cpd:DSS
Number Of Scans:128
Dummy Scans:4
Acquisition Time:1.24 s
Relaxation Delay:2 s
Spectral Width:16.4533 ppm
Num Data Points Acquired:32768
Line Broadening:0.3 Hz
Baseline Correction Method:Polynomial order 5