Potential Interacting Proteins For AMP-activated Protein Kinase α2 Revealed By Bacterial Two-Hybrid Screening

AMP-activated protein kinase (AMPK) is a ubiquitous protein kinase in all eukaryotes that regulates cellular metabolism and energy demand. Once activated AMPK phosphorylates a number of downstream substrates, the overall effect of which is to switch-off ATP-consuming (anabolic) pathways, and switch-on ATP-generating (catabolic) pathways, so called"cellular energy regulator". AMPK acts as a crucial component in energy metabolism not only regulating enzymes involved in glycometabolism, lipometabolism and protein metabolism, but also mediating genetic transcription and translation, which shows that AMPK plays a key role in maintaining energy balance within cells. Hypoxia, a principle cause of affecting human health and labor efficiency in high altitude, causes diminution of energy of mitochondria with disorders of oxidative phosphorylation. Maintaining energy balance has been a key issue of relieving anoxic injury. We postulate that AMPK may be an important factor in regulating energy homeostasis during hypoxia. The brain has a high metabolic rate and is sensitive to changes in the supply of energy and oxygen, which determines that it is a vulnerable organ. Therefore, it would contribute to looking for approaches to enhance hypoxia acclimatization that studying biological role and regulatory mechanism of AMPK in the brain. AMPK might be a potential novel target for countering anoxic injury.Biological processes are executed and controlled by proteins. Their intrinsic biochemical and/or catalytic activities are, to large extent, modulated by protein-protein interactions. Function can therefore be readily inferred from the identification of interaction partners. It is necessary for us to look for proteins interacting with AMPK to facilitate its functional annotation.AMPK is a heterotrimeric complex consisting of a catalytic subunit (α) and two regulatory subunits (βandγ). Eachαandβsubunit is encoded by two genes (α1 andα2 orβ1 andβ2), whereas theγsubunit is encoded by three genes (γ1,γ2, andγ3). Theαcatalytic subunit acts as a distinctive structure, so proteins that interact with it represent, to a certain extent, proteins that interact specifically with AMPK. Considering that theα2 catalytic subunit is more highly expressed thanα1 catalytic subunit in neurons, this study choosedα2 catalytic subunit as the bait to screen a fetal brain cDNA library by bacterial two-hybrid system for its interacting proteins. The study may be a foundation for further understanding the role of AMPK in the regulation of energy homeostasis in the brain, and provide new insight into elucidating mechanism of AMPK in the brain during anoxic adaptation.The following experiments were performed in this study:1. Cloning of AMPKα2 subunit coding sequence and construction of bacterial two-hybrid bait plasmid as well as identification.The coding sequence fragment of AMPKα2 subunit amplified by PCR was fused in frame with theλcI of pBT vector to form recombinant pBT-AMPKα2 plasmid. Restriction digestion and sequence analysis showed that the AMPKα2 coding sequence was correctly inserted into pBT with a right reading frame. Recombinant pBT-AMPKα2 plasmid was constructed successfully, namely bait plasmid.2. Expression of bait plasmid and identification of self-activation.(1) The bait plasmid pBT-AMPKα2 was transformed into XL1-Blue MR competent cells and induced by IPTG at 37℃incubation temperature. Cell lysates (whole cell extracts, supernatant fluid and sediment) were resolved by SDS-PAGE, but AMPKα2-λcI fusion protein could not be found, while Western blotting detected the fusion protein bands, and moreover, the AMPKα2-λcI fusion protein was expressed in two forms, soluble protein as well as incusion body. The former was unstabled and inclined to be broken down producing AMPKα2 andλcI proteins, on the contrary, the latter was stabled and not broken down.(2) XL1-Blue MR reporter strain was cotransformed with the recombinant pBT-AMPKα2 plasmid and the empty pTRG vector, and planted on Selective Screening Medium (3-AT) plates and Nonselective Screening Medium (no 3-AT) plates. In addition, cotransformation of pBT empty vector and pTRG-Gal11P was used as negative control in this experiment. The results showed that cotransformants in the negative control were grown on no 3-AT plates, while none was obtained on 3-AT plates, which verified that 3-AT selection was working properly. Colonies cotransformed with recombinant pBT-AMPKα2 and empty pTRG vector were obtained on no 3-AT plates, while none grew on 3-AT plates. This indicated that the recombinant plasmid pBT-AMPKα2 was incapable of activation of the reporter cassette in the absence of an interaction partner and could be used as"bait plasmid"in the bacterial two-hybrid system for screening.3. cDNA library amplification and screening of AMPKα2 subunit interacting proteins as well as identification.(1) cDNA library was amplified according to the operating manual. The amplified library was used to perform gradient dilution of culture for plating. 10 colonies were selected at random and their plasmids DNA were purified for DNA sequence analysis. Sequencing results showed that each of 10 clones contained a different sequence. Therefore, there was no bias in the amplified library, that is to say, the diversity of the amplified library remained.(2) For the pilot library cotransformation, transformed 100μl XL1-Blue MR reporter strain competent cells with 50ng of the recombinant pBT-AMPKα2 plasmid plus 50ng of the pTRG target cDNA library DNA by means of CaCl2 chemical method. Used the results of this pilot cotransformation to calculate the number of cotransformation reactions required to achieve library coverage. Then performed a large-scale cotransformation of the 500μl reporter strain with 200ng recombinant pBT-AMPKα2 plasmid plus 200ng the pTRG cDNA library DNA, using the calculated number of cotransformation reactions. After plating the library screen cotransformants on 3-AT plates and enrichment of putative interactors, 592 clones expressing HIS3 reporter gene were obtained by the primary screening.(3) Picked clones from primary screening and repatched them onto Dual Selective Screening Medium containing 3-AT and streptomycin (Strep) for the secondary screening. 20 false positive clones were eliminated because they were not grown on Dual Selective Screening Medium, however, the remaining clones expressing both HIS3 and aadA reporter genes were obtained by the secondary screening.(4) Picked 20 early-arising putative positive clones from the secondary screening, used the plasmid DNA isolated to transform XL1-Blue MRF′Kan competent cells, and plated the transformants on LB-tetracycline (LB-Ter) agar plates for purification of plasmid DNA from the 3-AT resistant colonies isolated. Then patched colonies from each plate onto each of two plates: one plate containing LB-chloramphenicol (LB-Cam) and the other containing LB-Ter for isolation of putative positive pTRG target from bait plasmid. The results showed that 1 was false positive clone and the remaining 19 were positive candidate clones.(5) Validation of 19 positive candidate clones by means of their retransformation into XL1-Blue MR reporter strain. Cotransformed the reporter strain respectively using each purified target plasmid paired with the recombinant pBT plasmid and paired with the empty pBT vector, and incubated on 3-AT Selective Screening Medium. The results showed that 9 clones were found to possess capability of specially interacting with AMPKα2, that is to say, real positive clones.(6) The plasmids of 9 positive clones were extracted, analyzed by DNA sequencing and their homologous proteins were researched in GenBank. Results showed that 7 AMPKα2 interacting partner proteins were identified, including phosphofructokinase, polyubiquitin, cytochrome c oxidase subunit I (COX I), heat shock protein 8 (HSP8), HLA-B-associated transcript 3 (BAT3) isoform 1, protein tyrosine phosphatase receptor type D (Ptprd) and islet-brain 1 (IB1). They were involved in glycolysis, protein degradation, mitochondrial electron transport chain and apoptosis being in on energy regulation directly or indirectly.