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In Situ Monitoring of p53 Protein and MDM2 Protein Interaction in Single Living Cells Using Single-Molecule Fluorescence Spectroscopy

Du, Zhixue (författare)
Shanghai Jiao Tong Univ, Sch Chem & Chem Engn, State Key Lab Met Matrix Composites, 800 Dongchuan Rd, Shanghai 200240, Peoples R China.
Yu, Jing (författare)
Shanghai Jiao Tong Univ, Sch Life Sci & Biotechnol, State Key Lab Microbial Metab, 800 Dongchuan Rd, Shanghai 200240, Peoples R China.
Li, Fucai (författare)
Shanghai Jiao Tong Univ, Sch Chem & Chem Engn, State Key Lab Met Matrix Composites, 800 Dongchuan Rd, Shanghai 200240, Peoples R China.
visa fler...
Deng, Liyun (författare)
Shanghai Jiao Tong Univ, Sch Chem & Chem Engn, State Key Lab Met Matrix Composites, 800 Dongchuan Rd, Shanghai 200240, Peoples R China.
Wu, Fang (författare)
Shanghai Jiao Tong Univ, Shanghai Ctr Syst Biomed, Minist Educ, Key Lab Syst Biomed, 800 Dongchuan Rd, Shanghai 200240, Peoples R China.
Huang, Xiangyi (författare)
Shanghai Jiao Tong Univ, Sch Chem & Chem Engn, State Key Lab Met Matrix Composites, 800 Dongchuan Rd, Shanghai 200240, Peoples R China.
Bergstrand, Jan (författare)
KTH,Experimentell biomolekylär fysik
Widengren, Jerker (författare)
KTH,Experimentell biomolekylär fysik
Dong, Chaoqing (författare)
Shanghai Jiao Tong Univ, Sch Chem & Chem Engn, State Key Lab Met Matrix Composites, 800 Dongchuan Rd, Shanghai 200240, Peoples R China.
Ren, Jicun (författare)
Shanghai Jiao Tong Univ, Sch Chem & Chem Engn, State Key Lab Met Matrix Composites, 800 Dongchuan Rd, Shanghai 200240, Peoples R China.
visa färre...
Shanghai Jiao Tong Univ, Sch Chem & Chem Engn, State Key Lab Met Matrix Composites, 800 Dongchuan Rd, Shanghai 200240, Peoples R China Shanghai Jiao Tong Univ, Sch Life Sci & Biotechnol, State Key Lab Microbial Metab, 800 Dongchuan Rd, Shanghai 200240, Peoples R China. (creator_code:org_t)
American Chemical Society (ACS), 2018
2018
Engelska.
Ingår i: Analytical Chemistry. - : American Chemical Society (ACS). - 0003-2700 .- 1520-6882. ; 90:10, s. 6144-6151
  • Tidskriftsartikel (refereegranskat)
Abstract Ämnesord
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  • Protein-protein interactions play a central role in signal transduction, transcription regulations, enzymatic activity, and protein synthesis. The p53 protein is a key transcription factor, and its activity is precisely regulated by the p53-MDM2 interaction. Although the p53-MDM2 interaction has been studied, it is still not clear how p53 structures and external factors influence the p53-MDM2 interaction in living cells. Here, we developed a direct method for monitoring the p53-MDM2 interaction in single living cells using single-molecule fluorescence cross-correlation spectroscopy with a microfluidic chip. First, we labeled p53 and MDM2 proteins with enhanced green fluorescent protein (EGFP) and mCherry, respectively, using lentivirus infection. We then designed various mutants covering the three main domains of p53 (tetramerization, transactivation, and DNA binding domains) and systematically studied effects of p53 protein primary, secondary, and quaternary structures on p53 MDM2 binding affinity in single living cells. We found that p53 dimers and tetramers can bind to MDM2, that the binding affinity of p53 tetramers is higher than that of p53 dimers, and that the affinity is closely correlated to the helicity of the p53 transactivation domain. The hot-spot mutation R175H in the DNA-binding domain reduced the binding of p53 to MDM2. Finally, we studied effects of inhibitors on p53-MDM2 interactions and dissociation dynamics of pS3-MDM2 complexes in single living cells. We found that inhibitors Nutlin 3 alpha and MI773 efficiently inhibited the pS3-MDM2 interaction, but RITA did not work in living cells. This study provides a direct way for quantifying the relationship between protein structure and protein protein interactions and evaluation of inhibitors in living cells.

Ämnesord

NATURAL SCIENCES  -- Chemical Sciences (hsv//eng)
NATURVETENSKAP  -- Kemi (hsv//swe)

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