TY - JOUR
T1 - A quantitative systems approach to define novel effects of tumour p53 mutations on binding oncoprotein mdm2
AU - Fuentes, Manuel
AU - Srivastava, Sanjeeva
AU - Gronenborn, Angela M.
AU - Labaer, Joshua
N1 - Funding Information:
Financial support to Manuel Fuentes from Health Institute Carlos III of Spain (ISCIII FIS‐ FEDER PI17/01930 & PI21/01545, CIBER‐ONC CB16/12/00400) as gratefully acknowledge. We also acknowledge Fondos FEDER (EU), Junta Castilla‐León (COVID19 grant COV20EDU/00187). The Proteomics Unit belongs to ProteoRed, PRB3‐ISCIII, supported by grant PT17/0019‐0023, of the PE I + D + I 2017‐2020, funded by ISCIII and FEDER.Financial support to Sanjeeva Srivastava from the Natural Sciences and Engineering Research Council (NSERC) of Canada is gratefully acknowledged. This work was supported in part by the Physical Sciences in Oncology Grant 5U54CA143907‐ 03. Authors acknowledge C. Boozer, Plexera USA and D. Larson (Draper, UT, USA) for technical support in SPRi; Rolfs A., Hu Y.; Barderas R., E. Hainsworth and N. Ramachan-dran, Harvard Institute of Proteomics, USA for cloning work and Lahav, G. for useful discussions. All our colleagues during our stay at Harvard Institute of Proteomics, Harvard Medical School, MA, USA.
Funding Information:
Funding: Financial support to Manuel Fuentes from Health Institute Carlos III of Spain (ISCIII FIS‐ FEDER PI17/01930 & PI21/01545, CIBER‐ONC CB16/12/00400) as gratefully acknowledge. We also acknowledge Fondos FEDER (EU), Junta Castilla‐León (COVID19 grant COV20EDU/00187). The Proteomics Unit belongs to ProteoRed, PRB3‐ISCIII, supported by grant PT17/0019‐0023, of the PE I + D + I 2017‐2020, funded by ISCIII and FEDER.Financial support to Sanjeeva Srivastava from the Natural Sciences and Engineering Research Council (NSERC) of Canada is gratefully acknowl‐ edged. This work was supported in part by the Physical Sciences in Oncology Grant 5U54CA143907‐ 03.
Publisher Copyright:
© 2021 by the authors. Licensee MDPI, Basel, Switzerland.
PY - 2022/1/1
Y1 - 2022/1/1
N2 - Understanding transient protein interactions biochemically at the proteome scale remains a long‐standing challenge. Current tools developed to study protein interactions in high‐throughput measure stable protein complexes and provide binary readouts; they do not elucidate dynamic and weak protein interactions in a proteome. The majority of protein interactions are transient and cover a wide range of affinities. Nucleic acid programmable protein arrays (NAPPA) are self‐assembling protein microarrays produced by freshly translating full‐length proteins in situ on the array surface. Herein, we have coupled NAPPA to surface plasmon resonance imaging (SPRi) to produce a novel label‐free platform that measures many protein interactions in real‐time allowing the determination of the KDs and rate constants. The developed novel NAPPA‐SPRi technique showed excellent abil-ity to study protein‐protein interactions of clinical mutants of p53 with its regulator MDM2. Fur-thermore, this method was employed to identify mutant p53 proteins insensitive to the drug nutlin‐ 3, currently in clinical practice, which usually disrupts the p53‐MDM2 interactions. Thus, significant differences in the interactions were observed for p53 mutants on the DNA binding domain (Arg‐ 273‐Cys, Arg‐273‐His, Arg‐248‐Glu, Arg‐280‐Lys), on the structural domain (His‐179‐Tyr, Cys‐176‐ Phe), on hydrophobic moieties in the DNA binding domain (Arg‐280‐Thr, Pro‐151‐Ser, Cys‐176‐ Phe) and hot spot mutants (Gly‐245‐Cys, Arg‐273‐Leu, Arg‐248‐Glu, Arg‐248‐Gly), which signifies the importance of point mutations on the MDM2 interaction and nutlin3 effect, even in molecular locations related to other protein activities.
AB - Understanding transient protein interactions biochemically at the proteome scale remains a long‐standing challenge. Current tools developed to study protein interactions in high‐throughput measure stable protein complexes and provide binary readouts; they do not elucidate dynamic and weak protein interactions in a proteome. The majority of protein interactions are transient and cover a wide range of affinities. Nucleic acid programmable protein arrays (NAPPA) are self‐assembling protein microarrays produced by freshly translating full‐length proteins in situ on the array surface. Herein, we have coupled NAPPA to surface plasmon resonance imaging (SPRi) to produce a novel label‐free platform that measures many protein interactions in real‐time allowing the determination of the KDs and rate constants. The developed novel NAPPA‐SPRi technique showed excellent abil-ity to study protein‐protein interactions of clinical mutants of p53 with its regulator MDM2. Fur-thermore, this method was employed to identify mutant p53 proteins insensitive to the drug nutlin‐ 3, currently in clinical practice, which usually disrupts the p53‐MDM2 interactions. Thus, significant differences in the interactions were observed for p53 mutants on the DNA binding domain (Arg‐ 273‐Cys, Arg‐273‐His, Arg‐248‐Glu, Arg‐280‐Lys), on the structural domain (His‐179‐Tyr, Cys‐176‐ Phe), on hydrophobic moieties in the DNA binding domain (Arg‐280‐Thr, Pro‐151‐Ser, Cys‐176‐ Phe) and hot spot mutants (Gly‐245‐Cys, Arg‐273‐Leu, Arg‐248‐Glu, Arg‐248‐Gly), which signifies the importance of point mutations on the MDM2 interaction and nutlin3 effect, even in molecular locations related to other protein activities.
KW - High‐throughput label‐free detection
KW - P53‐mdm2 interaction
KW - Protein microarrays
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U2 - 10.3390/ijms23010053
DO - 10.3390/ijms23010053
M3 - Article
C2 - 35008477
AN - SCOPUS:85121381002
VL - 23
JO - International Journal of Molecular Sciences
JF - International Journal of Molecular Sciences
SN - 1661-6596
IS - 1
M1 - 53
ER -