Recent advances in proximity-based labeling methods for interactome mapping

Trinkle-Mulcahy, Laura

doi:10.12688/f1000research.16903.1

Cited by 130 publications

(118 citation statements)

References 60 publications

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“…Subsequently, streptavidin-purified proteins can be identified and quantified by mass spectrometry (Figure 1B). When appropriate controls and mass spectrometric quantification are employed (as discussed elsewhere - (18)(19)(20)), PDB-MS can report on specific proximity relationships. It is important to keep in mind that what PDB-MS provides is a qualitative metric of the relative proximity between bait and prey and cannot explicitly determine whether the detected proteins are physically interacting (either directly or indirectly), or whether they are simply localized to the same area.…”

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confidence: 99%

“…Since the introduction of the first PDB-MS approach (reviewed in (18,19,21,22)), a growing number of enzymes -that largely fall into two groups: the biotin protein ligases and the peroxidases -as well as additional tools and experimental designs have made the strategy a flexible mainstay of interaction and organellar proteomics. Here, we will focus on the molecular basis for the main proximitydependent biotinylation approaches and on the development of distinct toolsets for the application of proximity dependent biotinylation to different biological questions.…”

Section: Downloaded Frommentioning

confidence: 99%

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Proximity Dependent Biotinylation: Key Enzymes and Adaptation to Proteomics Approaches

Samavarchi-Tehrani

Samson

Gingras

2020

Molecular & Cellular Proteomics

146

150

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The study of protein subcellular distribution, their assembly into complexes and the set of proteins with which they interact with is essential to our understanding of fundamental biological processes. Complementary to traditional assays, proximity-dependent biotinylation (PDB) approaches coupled with mass spectrometry (such as BioID or APEX) have emerged as powerful techniques to study proximal protein interactions and the subcellular proteome in the context of living cells and organisms. Since their introduction in 2012, PDB approaches have been used in an increasing number of studies and the enzymes themselves have been subjected to intensive optimization. How these enzymes have been optimized and considerations for their use in proteomics experiments are important questions. Here, we review the structural diversity and mechanisms of the two main classes of PDB enzymes: the biotin protein ligases (BioID) and the peroxidases (APEX). We describe the engineering of these enzymes for PDB and review emerging applications, including the development of PDB for coincidence detection (split-PDB). Lastly, we briefly review enzyme selection and experimental design guidelines and reflect on the labeling chemistries and their implication for data interpretation.

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Section: Downloaded Frommentioning

confidence: 99%

Section: Downloaded Frommentioning

confidence: 99%

Proximity Dependent Biotinylation: Key Enzymes and Adaptation to Proteomics Approaches

Samavarchi-Tehrani

Samson

Gingras

2020

Molecular & Cellular Proteomics

146

150

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“…In recent years, PBL has emerged as a powerful complementary approach to classic affinity purification of multiprotein complexes in mapping of protein-protein interactions [23] . By fusing proteins of interest to enzymes that generate reactive molecules, most commonly biotin, adjacent proteins are covalently labeled so that they can be isolated and identified [22] .…”

Section: Resultsmentioning

confidence: 99%

CBRPP: a new RNA-centric method to study RNA-protein interactions

Liu

Cao

et al. 2020

Preprint

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0RNA-protein interactions play essential roles in tuning gene expression at RNA level and 1 1 modulating the function of proteins. Abnormal RNA-protein interactions lead to cell dysfunction 1 2 and human diseases. Therefore, mapping networks of RNA-protein interactions is crucial for 1 3 understanding cellular mechanism and pathogenesis of diseases. Different practical protein-centric 1 4 methods for studying RNA-protein interactions has been reported, but few RNA-centric methods 1 5 exist. Here, we developed CRISPR-based RNA proximity proteomics (CBRPP), a new 1 6RNA-centric method to identify proteins associated with the target RNA in native cellular context 1 7 without cross-linking or RNA manipulation in vitro. CBRPP is based on a fusion of dCas13 and 1 8 proximity-based labeling (PBL) enzyme. dCas13 can deliver PBL enzyme to the target RNA with 1 9 high specificity, while PBL enzyme labels the surrounding proteins of the target RNA, which are 2 0 then identified by mass spectrometry. 2 1 Keywords: RNA-protein interactions, dPspCas13b, dRfxCas13d, APEX2, TurboID, BASU, 2 2 BioID2, CBRPP 2 3 Introduction 2 4RNA is bound to protein from birth to death. RNA-binding proteins (RBPs) play a pivotal role 2 5 in a wide range of biological processes, including RNA transcription, processing, modification, 2 6 transport, translation and stabilization [1][2][3][4] . RNAs, in turn, influence proteins expression, 2 7 localization and interactions with other proteins [5][6][7] . Aberrant RNA-protein interactions are related 2 8to cellular dysfunction and human diseases [3, 8, 9] . Therefore, mapping networks of RNA-protein 2 9interactions is of great importance for understanding many cellular biological processes. 0Based on the type of molecule they start with, methods for studying RNA-protein interactions 3 1 are classified into protein-centric methods and RNA-centric methods [10] . Protein-centric methods 3 2 start with a protein of interest and study RNAs that interact with that protein. Since proteins are 3 3 easily purified with antibodies, many protein-centric methods, such as cross-linking 3 4 immunoprecipitation (CLIP)-seq [11] and RNA immunoprecipitation (RIP)-seq [12] , are available and 3 5 practical. Conversely, RNA-centric methods start with an RNA of interest and focus on proteins 3 6 that bind it. Most approaches use biotinylated RNA [13] , aptamer-tagged RNA [14] , peptide nucleic 3 7 acid [15] and antisense probe [16][17][18][19][20] for purification of RNA-protein complexes to identify proteins 3 8 that associate with the target RNA, however these methods often require RNA manipulation in 3 9 vitro and miss transient or weak interactions. Meanwhile, compared with protein-centric methods, 4 0 there are few robust RNA-centric methods. 4 1 In this paper, by combining the power of CRISPR-Cas13 [21] and proximity-based labeling (PBL) 4 2 technique [22] , we developed CBRPP (CRISPR-based RNA proximity proteomics), a new 4 3 RNA-centric method to identify proteins associated with the target RNA in native c...

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“…Promiscuous biotin ligases have emerged as powerful tools for proximity-based protein identification in live cells (48). To date, the original E. coli BirA* (BioID) has been employed by four studies in Plasmodium spp ., including two that targeted BioID to the PV (49–52).…”

Section: Discussionmentioning

confidence: 99%

EXP1 is required for organization of the intraerythrocytic malaria parasite vacuole

Nessel

Beck

Rayatpisheh³

et al. 2019

Preprint

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word count: 250 22 23 24 Importance word count: 149 25 26 Abstract 27Intraerythrocytic malaria parasites reside within a parasitophorous vacuole membrane (PVM) 28 that closely overlays the parasite plasma membrane (PPM) and constitutes the barrier between 29 parasite and host compartments. The PVM is the site of several essential transport activities but 30 the basis for organization of this membrane system is unknown. We utilized the second-31 generation promiscuous biotin ligase BioID2 fused to EXP2 or HSP101 to probe the content of 32 the PVM, identifying known and novel candidate PVM proteins. Among the best represented 33 hits were members of a group of single-pass integral membrane proteins that constitute a major 34 component of the PVM proteome but whose function remains unclear. We investigated the 35 function of EXP1, the longest known member of this group, by adapting a CRISPR/Cpf1 36 genome editing system to install the TetR-DOZI-aptamers system for conditional translational 37 control. EXP1 knockdown was essential for intraerythrocytic development and accompanied by 38 profound changes in vacuole ultrastructure, including increased separation of the PVM and 39 PPM and formation of abnormal membrane structures in the enlarged vacuole lumen. While 40 previous in vitro studies indicated EXP1 possesses glutathione S-transferase activity, a mutant 41 version of EXP1 lacking a residue important for this activity in vitro still provides substantial 42 rescue of endogenous exp1 knockdown in vivo. Intriguingly, while activity of the Plasmodium 43 translocon of exported proteins was not impacted by depletion of EXP1, the distribution of the 44 translocon pore-forming protein EXP2 was substantially altered. Collectively, our results reveal 45 a novel PVM defect that indicates a critical role for EXP1 in maintaining proper PVM 46 organization. 47 48 Importance 49 Like other obligate intracellular apicomplexans, blood-stage malaria parasites reside within a 50 membrane-bound compartment inside the erythrocyte known as the parasitophorous vacuole. 51Although the vacuole is the site of several transport activities essential to parasite survival, little 52 is known about its organization. To explore vacuole biology, we adopted recently developed 53 proteomic (BioID2) and genetic (CRISPR/Cpf1) tools for use in Plasmodium falciparum, which 54 allowed us to query the function of the prototypical vacuole membrane protein EXP1. 55Knockdown of EXP1 showed that a previously reported glutathione S-transferase activity cannot 56 fully account for the essential function(s) of EXP1 and revealed a novel role for this protein in 57 maintaining normal vacuole morphology and PVM protein arrangement. Our results provide new 58 insight into vacuole organization and illustrate the power of BioID2 and Cpf1 (which utilizes a T-59 rich PAM uniquely suited to the P. falciparum genome) for proximity protein identification and 60 genome editing in P. falciparum. 61 62 65principal barrier between the parasite and its host cell (1). Durin...

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Recent advances in proximity-based labeling methods for interactome mapping

Cited by 130 publications

References 60 publications

Proximity Dependent Biotinylation: Key Enzymes and Adaptation to Proteomics Approaches

Proximity Dependent Biotinylation: Key Enzymes and Adaptation to Proteomics Approaches

CBRPP: a new RNA-centric method to study RNA-protein interactions

EXP1 is required for organization of the intraerythrocytic malaria parasite vacuole

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