In Vivo Detection of Metabolic Fluctuations in Real Time Using the NanoBiT Technology Based on PII Signalling Protein Interactions
Abstract
:1. Introduction
2. Results
2.1. Establishment of PII-Based In Vivo NanoBiT Sensors
2.2. Monitoring Metabolic Fluctuations Using the PII-NAGK Sensor
2.3. Monitoring Metabolic Fluctuations Using the PII-PipX Sensor
2.4. Monitoring Metabolic Response upon Complete Nutrient Deprivation
3. Discussion
4. Materials and Methods
Cultivation of NanoBiT Expressing Cells and Luminescence Measurement
5. Conclusions
Author Contributions
Funding
Data Availability Statement
Acknowledgments
Conflicts of Interest
References
- Nooren, I.M.; Thornton, J.M. Diversity of protein–protein interactions. EMBO J. 2003, 22, 3486–3492. [Google Scholar] [CrossRef] [PubMed]
- Keskin, O.; Tuncbag, N.; Gursoy, A. Predicting protein–protein interactions from the molecular to the proteome level. Chem. Rev. 2016, 116, 4884–4909. [Google Scholar] [CrossRef] [PubMed]
- Liddington, R.C. Structural basis of protein-protein interactions. Protein-Protein Interact. Methods Appl. 2004, 261, 3–14. [Google Scholar]
- Keskin, O.; Gursoy, A.; Ma, B.; Nussinov, R. Principles of protein− protein interactions: What are the preferred ways for proteins to interact? Chem. Rev. 2008, 108, 1225–1244. [Google Scholar] [CrossRef]
- Blaszczak, E.; Lazarewicz, N.; Sudevan, A.; Wysocki, R.; Rabut, G. Protein-fragment complementation assays for large-scale analysis of protein–protein interactions. Biochem. Soc. Trans. 2021, 49, 1337–1348. [Google Scholar] [CrossRef]
- Verhoef, L.G.; Mattioli, M.; Ricci, F.; Li, Y.-C.; Wade, M. Multiplex detection of protein–protein interactions using a next generation luciferase reporter. Biochim. Biophys. Acta (BBA)-Mol. Cell Res. 2016, 1863, 284–292. [Google Scholar] [CrossRef]
- Westerhausen, S.; Nowak, M.; Torres-Vargas, C.E.; Bilitewski, U.; Bohn, E.; Grin, I.; Wagner, S. A NanoLuc luciferase-based assay enabling the real-time analysis of protein secretion and injection by bacterial type III secretion systems. Mol. Microbiol. 2020, 113, 1240–1254. [Google Scholar] [CrossRef] [PubMed]
- Thalwieser, Z.; Király, N.; Fonódi, M.; Csortos, C.; Boratkó, A. Protein phosphatase 2A–mediated flotillin-1 dephosphorylation up-regulates endothelial cell migration and angiogenesis regulation. J. Biol. Chem. 2019, 294, 20196–20206. [Google Scholar] [CrossRef]
- Yang, X.; Liu, L.; Hao, Y.; So, E.; Emami, S.S.; Zhang, D.; Gong, Y.; Sheth, P.M.; Wang, Y. A bioluminescent biosensor for quantifying the interaction of SARS-CoV-2 and its receptor ACE2 in cells and in vitro. Viruses 2021, 13, 1055. [Google Scholar] [CrossRef]
- Tsang, T.F.; Qiu, Y.; Lin, L.; Ye, J.; Ma, C.; Yang, X. Simple method for studying in vitro protein–protein interactions based on protein complementation and its application in drug screening targeting bacterial transcription. ACS Infect. Dis. 2019, 5, 521–527. [Google Scholar] [CrossRef]
- Wood, A.; Irving, S.E.; Bennison, D.J.; Corrigan, R.M. The (p)ppGpp-binding GTPase Era promotes rRNA processing and cold adaptation in Staphylococcus aureus. PLoS Genet. 2019, 15, e1008346. [Google Scholar] [CrossRef]
- Pipchuk, A.; Yang, X. Using Biosensors to Study Protein–Protein Interaction in the Hippo Pathway. Front. Cell Dev. Biol. 2021, 9, 660137. [Google Scholar] [CrossRef]
- Cooley, R.; Kara, N.; Hui, N.S.; Tart, J.; Roustan, C.; George, R.; Hancock, D.C.; Binkowski, B.F.; Wood, K.V.; Ismail, M. Development of a cell-free split-luciferase biochemical assay as a tool for screening for inhibitors of challenging protein-protein interaction targets. Wellcome Open Res. 2020, 5, 20. [Google Scholar] [CrossRef] [PubMed]
- Sicking, M.; Jung, M.; Lang, S. Lights, Camera, Interaction: Studying Protein–Protein Interactions of the ER Protein Translocase in Living Cells. Int. J. Mol. Sci. 2021, 22, 10358. [Google Scholar] [CrossRef] [PubMed]
- Kashima, D.; Kageoka, M.; Kimura, Y.; Horikawa, M.; Miura, M.; Nakakido, M.; Tsumoto, K.; Nagamune, T.; Kawahara, M. A Novel Cell-Based Intracellular Protein–Protein Interaction Detection Platform (SOLIS) for Multimodality Screening. ACS Synth. Biol. 2021, 10, 990–999. [Google Scholar] [CrossRef] [PubMed]
- Paiva, A.M.O.; Friggen, A.H.; Qin, L.; Douwes, R.; Dame, R.T.; Smits, W.K. The bacterial chromatin protein HupA can remodel DNA and associates with the nucleoid in Clostridium difficile. J. Mol. Biol. 2019, 431, 653–672. [Google Scholar] [CrossRef] [PubMed]
- Bardelang, P.; Murray, E.J.; Blower, I.; Zandomeneghi, S.; Goode, A.; Hussain, R.; Kumari, D.; Siligardi, G.; Inoue, K.; Luckett, J. Conformational analysis and interaction of the Staphylococcus aureus transmembrane peptidase AgrB with its AgrD propeptide substrate. Front. Chem. 2023, 11, 1113885. [Google Scholar] [CrossRef] [PubMed]
- Forchhammer, K.; Selim, K.A.; Huergo, L.F. New views on PII signaling: From nitrogen sensing to global metabolic control. Trends Microbiol. 2022, 30, 722–735. [Google Scholar] [CrossRef]
- Forchhammer, K.; Lüddecke, J. Sensory properties of the PII signalling protein family. FEBS J. 2016, 283, 425–437. [Google Scholar] [CrossRef]
- Lüddecke, J.; Forchhammer, K. From PII signaling to metabolite sensing: A novel 2-oxoglutarate sensor that details PII-NAGK complex formation. PLoS ONE 2013, 8, e83181. [Google Scholar]
- Chen, H.L.; Bernard, C.S.; Hubert, P.; My, L.; Zhang, C.C. Fluorescence resonance energy transfer based on interaction of PII and PipX proteins provides a robust and specific biosensor for 2-oxoglutarate, a central metabolite and a signalling molecule. FEBS J. 2014, 281, 1241–1255. [Google Scholar] [CrossRef]
- Remy, I.; Michnick, S.W. Application of protein-fragment complementation assays in cell biology. Biotechniques 2007, 42, 137–145. [Google Scholar] [CrossRef]
- Lüddecke, J.; Francois, L.; Spät, P.; Watzer, B.; Chilczuk, T.; Poschet, G.; Hell, R.; Radlwimmer, B.; Forchhammer, K. PII protein-derived FRET sensors for quantification and live-cell imaging of 2-oxoglutarate. Sci. Rep. 2017, 7, 1437. [Google Scholar] [CrossRef]
- Rozbeh, R.; Forchhammer, K. Split NanoLuc technology allows quantitation of interactions between PII protein and its receptors with unprecedented sensitivity and reveals transient interactions. Sci. Rep. 2021, 11, 12535. [Google Scholar] [CrossRef] [PubMed]
- Fokina, O.; Herrmann, C.; Forchhammer, K. Signal-transduction protein PII from Synechococcus elongatus PCC 7942 senses low adenylate energy charge in vitro. Biochem. J. 2011, 440, 147–156. [Google Scholar] [CrossRef] [PubMed]
- Zeth, K.; Fokina, O.; Forchhammer, K. Structural basis and target-specific modulation of ADP sensing by the Synechococcus elongatus PII signaling protein. J. Biol. Chem. 2014, 289, 8960–8972. [Google Scholar] [CrossRef] [PubMed]
- Radchenko, M.V.; Thornton, J.; Merrick, M. Association and dissociation of the GlnK–AmtB complex in response to cellular nitrogen status can occur in the absence of GlnK post-translational modification. Front. Microbiol. 2014, 5, 124255. [Google Scholar] [CrossRef] [PubMed]
- Yuan, J.; Doucette, C.D.; Fowler, W.U.; Feng, X.J.; Piazza, M.; Rabitz, H.A.; Wingreen, N.S.; Rabinowitz, J.D. Metabolomics-driven quantitative analysis of ammonia assimilation in E. coli. Mol. Syst. Biol. 2009, 5, 302. [Google Scholar] [CrossRef] [PubMed]
- Yuan, J.; Bennett, B.D.; Rabinowitz, J.D. Kinetic flux profiling for quantitation of cellular metabolic fluxes. Nat. Protoc. 2008, 3, 1328–1340. [Google Scholar] [CrossRef] [PubMed]
- Merck. pACYCDuet™-1 DNA—Novagen; Merck: Darmstadt, Germany, 2024. [Google Scholar]
- Addgene. Gibson Assembly Cloning; Addgene: Watertown, MA, USA, 2024. [Google Scholar]
- Bertani, G. Studies on lysogenesis I: The mode of phage liberation by lysogenic Escherichia coli. J. Bacteriol. 1951, 62, 293–300. [Google Scholar] [CrossRef] [PubMed]
- Studier, F.W.; Daegelen, P.; Lenski, R.E.; Maslov, S.; Kim, J.F. Understanding the differences between genome sequences of Escherichia coli B strains REL606 and BL21 (DE3) and comparison of the E. coli B and K-12 genomes. J. Mol. Biol. 2009, 394, 653–680. [Google Scholar] [CrossRef] [PubMed]
(−IPTG) | (+IPTG) | |||
---|---|---|---|---|
Constructs | RLU Signal | % | RLU Signal | % |
PII-FL | 4,721,437 ± 623,462 | 100 | 68,198,243 ± 7,174,184 | 100 |
PII-LgBiT-NAGK-SmBiT | 422,034 ± 81,375 | 9 | 6,072,362 ± 545,005 | 9 |
PII(S49E)-LgBiT-NAGK-SmBiT | 22,902 ± 2685 | 0.5 | nm | nm |
PII-LgBiT | 254 ± 42 | 0.005 | nm | nm |
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Rozbeh, R.; Forchhammer, K. In Vivo Detection of Metabolic Fluctuations in Real Time Using the NanoBiT Technology Based on PII Signalling Protein Interactions. Int. J. Mol. Sci. 2024, 25, 3409. https://doi.org/10.3390/ijms25063409
Rozbeh R, Forchhammer K. In Vivo Detection of Metabolic Fluctuations in Real Time Using the NanoBiT Technology Based on PII Signalling Protein Interactions. International Journal of Molecular Sciences. 2024; 25(6):3409. https://doi.org/10.3390/ijms25063409
Chicago/Turabian StyleRozbeh, Rokhsareh, and Karl Forchhammer. 2024. "In Vivo Detection of Metabolic Fluctuations in Real Time Using the NanoBiT Technology Based on PII Signalling Protein Interactions" International Journal of Molecular Sciences 25, no. 6: 3409. https://doi.org/10.3390/ijms25063409