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A proximity labeling method for protein-protein interactions on cell membrane

ABSTRACT: We introduce antigen-antibody proximity labeling (AAPL), a novel method to map the antigen interaction sites as well as interactors of antibody-targeted proteins. As a proof of concept, AAPL was demonstrated using sodium/potassium transporting ATPase (ATP1A1) and epidermal growth factor receptor 2 (ERBB2)-specific antibodies that were modified with an Fe(III) catalytic probe. Once bound to their target proteins, Fe(III)-induced catalytic oxidation occurred in proximity of the antigen’s epitope. Oxidative proteomic analysis was then used to determine the degree of oxidation, the site of oxidation within the targeted antigen, and the interacting proteins that were in close proximity to the targeted antigen. An AAPL score was generated for each protein yielding the specificity of the oxidation and proximity of the interacting protein to the target antigen. As a final demonstration of its utility, the AAPL approach was applied to map the interactors of liver-intestine-cadherin (CDH17) in colon cancer cells.

INSTRUMENT(S): Orbitrap Fusion Lumos

ORGANISM(S): Homo Sapiens (human)

SUBMITTER: Qiongyu Li

LAB HEAD: Carlito B Lebrilla

PROVIDER: PXD033466 | Pride | 2022-08-12

REPOSITORIES: Pride

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A proximity labeling method for protein-protein interactions on cell membrane.

Li Qiongyu Q Xie Yixuan Y Rice Rachel R Maverakis Emanual E Lebrilla Carlito B CB

Chemical science 20220430 20

Antibodies targeting specific antigens are widely utilized in biological research to investigate protein interactions or to quantify target antigens. Here, we introduce antigen-antibody proximity labeling (AAPL), a novel method to map the antigen interaction sites as well as interactors of antibody-targeted proteins. As a proof of concept, AAPL was demonstrated using sodium/potassium transporting ATPase (ATP1A1) and epidermal growth factor receptor 2 (ERBB2)-specific antibodies that were modifie ...[more]

PMID: 35685794

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Project description:Mahadevan2006 - Genome-scale metabolic network of Geobacter sulfurreducens (iRM588) This model is described in the article: Characterization of metabolism in the Fe(III)-reducing organism Geobacter sulfurreducens by constraint-based modeling. Mahadevan R, Bond DR, Butler JE, Esteve-Nuñez A, Coppi MV, Palsson BO, Schilling CH, Lovley DR. Appl. Environ. Microbiol. 2006 Feb; 72(2): 1558-1568 Abstract: Geobacter sulfurreducens is a well-studied representative of the Geobacteraceae, which play a critical role in organic matter oxidation coupled to Fe(III) reduction, bioremediation of groundwater contaminated with organics or metals, and electricity production from waste organic matter. In order to investigate G. sulfurreducens central metabolism and electron transport, a metabolic model which integrated genome-based predictions with available genetic and physiological data was developed via the constraint-based modeling approach. Evaluation of the rates of proton production and consumption in the extracellular and cytoplasmic compartments revealed that energy conservation with extracellular electron acceptors, such as Fe(III), was limited relative to that associated with intracellular acceptors. This limitation was attributed to lack of cytoplasmic proton consumption during reduction of extracellular electron acceptors. Model-based analysis of the metabolic cost of producing an extracellular electron shuttle to promote electron transfer to insoluble Fe(III) oxides demonstrated why Geobacter species, which do not produce shuttles, have an energetic advantage over shuttle-producing Fe(III) reducers in subsurface environments. In silico analysis also revealed that the metabolic network of G. sulfurreducens could synthesize amino acids more efficiently than that of Escherichia coli due to the presence of a pyruvate-ferredoxin oxidoreductase, which catalyzes synthesis of pyruvate from acetate and carbon dioxide in a single step. In silico phenotypic analysis of deletion mutants demonstrated the capability of the model to explore the flexibility of G. sulfurreducens central metabolism and correctly predict mutant phenotypes. These results demonstrate that iterative modeling coupled with experimentation can accelerate the understanding of the physiology of poorly studied but environmentally relevant organisms and may help optimize their practical applications. This model is hosted on BioModels Database and identified by: MODEL1507180000. To cite BioModels Database, please use: BioModels Database: An enhanced, curated and annotated resource for published quantitative kinetic models. 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.

Project description:Previous studies demonstrated that an outer membrane c-type cytochrome, OmcB (GSU_2737), was involved in Fe(III) reduction in Geobacter sulfurreducens. An OmcB-deficient mutant was greatly impaired in its ability to reduce both soluble and insoluble Fe(III). Reintroducing omcB restored the capacity for Fe(III) reduction at a level proportional to the level of OmcB production. Here, we report that the OmcB-deficient mutant gradually adapted to grow on soluble Fe(III) but not insoluble Fe(III). The adapted OmcB-deficient mutant reduced soluble Fe(III) at a rate comparable to that of the wild type, but the cell yield of the mutant was only ca. 60% of that of the wild type under steady-state culturing conditions. Analysis of proteins and transcript levels demonstrated that expression of several membrane-associated cytochromes was higher in the adapted mutant than in the wild type. Further comparison of transcript levels during steady-state growth on Fe(III) citrate with a whole-genome DNA microarray revealed a significant shift in gene expression in an apparent attempt to adapt metabolism to the impaired electron transport to Fe(III). These results demonstrate that, although there are many other membrane-bound c-type cytochromes in G. sulfurreducens, increased expression of these cytochromes cannot completely compensate for the loss of OmcB. The concept that outer membrane cytochromes are promiscuous reductases that are interchangeable in function appears to be incorrect. Furthermore, the results indicate that there may be different mechanisms for electron transfer to soluble Fe(III) and insoluble Fe(III) oxides in G. sulfurreducens, which emphasizes the importance of studying electron transport to the environmentally relevant Fe(III) oxides. (From Leang C, Adams LA, Chin KJ, Nevin KP, Methé BA, Webster J, Sharma ML, Lovley DR. Adaptation to disruption of the electron transfer pathway for Fe(III) reduction in Geobacter sulfurreducens. J Bacteriol. 2005 Sep;187(17):5918-26.) Keywords: Geobacter, gene expression, genetic modification

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