Summary of Study ST001375
This data is available at the NIH Common Fund's National Metabolomics Data Repository (NMDR) website, the Metabolomics Workbench, https://www.metabolomicsworkbench.org, 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.
See: https://www.metabolomicsworkbench.org/about/howtocite.php
| Study ID | ST001375 |
| Study Title | Fructosamine-3-kinase (FN3K) KO in HepG2 liver cancer cells |
| Study Summary | Fructosamine-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. |
| Institute | University of Georgia |
| Department | Complex Carbohydrate Research Center |
| Laboratory | Edison |
| Last Name | Colonna |
| First Name | Maxwell |
| Address | 315 Riverbend Rd, Athens, GA 30602 |
| maxwellbaca@uga.edu | |
| Phone | 7065420257 |
| Submit Date | 2020-05-08 |
| Num Groups | 2 |
| Publications | A redox-active switch in Fructosamine-3-kinases expands the regulatory repertoire of the protein kinase super-family |
| Raw Data Available | Yes |
| Raw Data File Type(s) | fid |
| Analysis Type Detail | NMR |
| Release Date | 2020-07-07 |
| Release Version | 1 |
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Project:
| 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 |
| Laboratory: | Kannan |
| Last Name: | Kannan |
| First Name: | Natarajan |
| Address: | B122 Life Sciences Bldg. University of Georgia Athens, GA 30602 |
| Email: | nkannan@uga.edu |
| Phone: | 706-542-1334 |
| 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:
| Subject ID: | SU001449 |
| Subject Type: | Cultured cells |
| Subject Species: | Homo sapiens |
| Taxonomy ID: | 9606 |
| Genotype Strain: | HepG2 |
Factors:
Subject type: Cultured cells; Subject species: Homo sapiens (Factor headings shown in green)
| mb_sample_id | local_sample_id | Genotype |
|---|---|---|
| SA100481 | FN3K_9 | FN3K KO |
| SA100482 | FN3K_10 | FN3K KO |
| SA100483 | FN3K_1 | FN3K KO |
| SA100484 | FN3K_7 | FN3K KO |
| SA100485 | FN3K_8 | FN3K KO |
| SA100486 | FN3K_6 | FN3K KO |
| SA100487 | FN3K_2 | FN3K KO |
| SA100488 | FN3K_4 | FN3K KO |
| SA100489 | FN3K_3 | FN3K KO |
| SA100490 | FN3K_5 | FN3K KO |
| SA100491 | WT_8 | WT |
| SA100492 | WT_9 | WT |
| SA100493 | WT_10 | WT |
| SA100494 | WT_7 | WT |
| SA100495 | WT_1 | WT |
| SA100496 | WT_2 | WT |
| SA100497 | WT_3 | WT |
| SA100498 | WT_4 | WT |
| SA100499 | WT_5 | WT |
| SA100500 | WT_6 | WT |
| Showing results 1 to 20 of 20 |
Collection:
| 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:
| 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:
| 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 |
| Units: | AU |
NMR:
| 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 |
| Temperature: | 27 |
| 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 |
