Dataset Information

AP-MS in CRISPR/Cas9 Utilizing Systems for Mapping Endogenous Protein Complexes

ABSTRACT: We combined clustered regularly interspaced short palindromic repeats/Cas9-mediated gene knockout technology with affinity purification using antibodies against endogenous proteins followed by mass spectrometry analysis, to sensitively and precisely detect protein-protein interactions in unaltered in vivo settings. Using this system, we analyzed endogenous neurofibromin-associated protein complexes.

INSTRUMENT(S): LTQ Orbitrap Velos

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Permanent Cell Line Cell, Cell Culture, Hela Cell

DISEASE(S): Cervix Carcinoma,Disease Free

SUBMITTER: Xu Li

LAB HEAD: Junjie Chen

PROVIDER: PXD005107 | Pride | 2017-03-15

REPOSITORIES: Pride

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Publications

Clustered, Regularly Interspaced Short Palindromic Repeats (CRISPR)/Cas9-coupled Affinity Purification/Mass Spectrometry Analysis Revealed a Novel Role of Neurofibromin in mTOR Signaling.

Li Xu X Gao Min M Choi Jong Min JM Kim Beom-Jun BJ Zhou Mao-Tian MT Chen Zhen Z Jain Antrix N AN Jung Sung Yun SY Yuan Jingsong J Wang Wenqi W Wang Yi Y Chen Junjie J

Molecular & cellular proteomics : MCP 20170207 4

Neurofibromin (NF1) is a well known tumor suppressor that is commonly mutated in cancer patients. It physically interacts with RAS and negatively regulates RAS GTPase activity. Despite the importance of NF1 in cancer, a high quality endogenous NF1 interactome has yet to be established. In this study, we combined clustered, regularly interspaced short palindromic repeats (CRISPR)/Cas9-mediated gene knock-out technology with affinity purification using ant ...[more]

PMID: 28174230

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Project description:Phillips2003 - The Mechanism of Ras GTPase Activation by Neurofibromin A mathematical model for Ras-GTP activation by neurofibromin and the kinetic rates of the relevant reactions. This model is described in the article: The mechanism of Ras GTPase activation by neurofibromin. Phillips RA, Hunter JL, Eccleston JF, Webb MR. Biochemistry 2003 Apr; 42(13): 3956-3965 Abstract: Individual rate constants have been determined for each step of the Ras.GTP hydrolysis mechanism, activated by neurofibromin. Fluorescence intensity and anisotropy stopped-flow measurements used the fluorescent GTP analogue, mantGTP (2'(3')-O-(N-methylanthraniloyl)GTP), to determine rate constants for binding and release of neurofibromin. Quenched flow measurements provided the kinetics of the hydrolytic cleavage step. The fluorescent phosphate sensor, MDCC-PBP was used to measure phosphate release kinetics. Phosphate-water oxygen exchange, using (18)O-substituted GTP and inorganic phosphate (P(i)), was used to determine the extent of reversal of the hydrolysis step and of P(i) binding. The data show that neurofibromin and P(i) dissociate from the NF1.Ras.GDP.P(i) complex with identical kinetics, which are 3-fold slower than the preceding cleavage step. A model is presented in which the P(i) release is associated with the change of Ras from "GTP" to "GDP" conformation. In this model, the conformation change on P(i) release causes the large change in affinity of neurofibromin, which then dissociates rapidly. This model is hosted on BioModels Database and identified by: BIOMD0000000692. To cite BioModels Database, please use: Chelliah V et al. BioModels: ten-year anniversary. Nucl. Acids Res. 2015, 43(Database issue):D542-8. To the extent possible under law, all copyright and related or neighbouring rights to this encoded model have been dedicated to the public domain worldwide. Please refer to CC0 Public Domain Dedication for more information.

Dataset Information

AP-MS in CRISPR/Cas9 Utilizing Systems for Mapping Endogenous Protein Complexes

Publications

Clustered, Regularly Interspaced Short Palindromic Repeats (CRISPR)/Cas9-coupled Affinity Purification/Mass Spectrometry Analysis Revealed a Novel Role of Neurofibromin in mTOR Signaling.

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