Proximity Dependent Biotinylation: Key Enzymes and Adaptation to Proteomics Approaches
2020; Elsevier BV; Volume: 19; Issue: 5 Linguagem: Inglês
10.1074/mcp.r120.001941
ISSN1535-9484
AutoresPayman Samavarchi‐Tehrani, Reuben Samson, Anne‐Claude Gingras,
Tópico(s)Vitamin D Research Studies
ResumoThe 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. 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. In eukaryotic cells, most processes and reactions are compartmentalized into organelles and other subcellular structures and often effected through the concerted action of molecular machines. 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However, this can be challenging when recovering structures or molecules that are difficult to solubilize or easily lose integrity through purification (12Mousson F. Kolkman A. Pijnappel W.W. Timmers H.T. Heck A.J. Quantitative proteomics reveals regulation of dynamic components within TATA-binding protein (TBP) transcription complexes.Mol. Cell. Proteomics. 2008; 7: 845-852Abstract Full Text Full Text PDF PubMed Scopus (63) Google Scholar, 13Wang X. Huang L. Identifying dynamic interactors of protein complexes by quantitative mass spectrometry.Mol. Cell. Proteomics. 2008; 7: 46-57Abstract Full Text Full Text PDF PubMed Scopus (151) Google Scholar). Strategies that attempt to overcome these limitations have been introduced in the past decade. Optimization of lysis and purification conditions has enabled the definition of interactomes for membrane proteins (14Babu M. Vlasblom J. Pu S. Guo X. Graham C. Bean B.D. Burston H.E. Vizeacoumar F.J. Snider J. Phanse S. Fong V. Tam Y.Y. Davey M. 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A pipeline for determining protein-protein interactions and proximities in the cellular milieu.Mol. Cell Proteomics. 2014; 13: 2824-2835Abstract Full Text Full Text PDF PubMed Scopus (27) Google Scholar, 17Yu C. Yang Y. Wang X. Guan S. Fang L. Liu F. Walters K.J. Kaiser P. Huang L. Characterization of Dynamic UbR-proteasome subcomplexes by in vivo cross-linking (X) assisted bimolecular tandem affinity purification (XBAP) and label-free quantitation.Mol. Cell Proteomics. 2016; 15: 2279-2292Abstract Full Text Full Text PDF PubMed Scopus (19) Google Scholar), though this may also increase the number of false positive interactors in some cases. In the past 8 years, alternative approaches have been introduced that instead bypass the requirement to maintain protein-protein interactions or organellar integrity during sample purification. Collectively, these are referred to as proximity-dependent biotinylation (PDB) 1The abbreviations used are:PDBproximity-dependent biotinylationAP-MSaffinity purification coupled to mass spectrometryAPXascorbate peroxidaseBARbiotinylation by antibody recognitionBirAbifunctional ligase/repressorBCCPbiotin-carboxylase cargo proteinBPLbiotin protein ligaseEMARSenzyme-mediated activation of radical sourcesERendoplasmic reticulumIDRintrinsically disordered regionivBioIDin vitro BioIDFKBPFK506-binding proteinFRBFKBP-rapamycin binding domainHRPhorseradish peroxidase CNHS-biotinN-hydroxysuccinimide-biotinPCAprotein-fragment complementation assayPDB-MSproximity-dependent biotinylation coupled to mass spectrometrySPPLATselective proteomic proximity labeling using tyramideSILACstable isotope labeling by amino acids in cell cultureTMTtandem mass tagiTRAQisobaric tag for relative and absolute quantification. 1The abbreviations used are:PDBproximity-dependent biotinylationAP-MSaffinity purification coupled to mass spectrometryAPXascorbate peroxidaseBARbiotinylation by antibody recognitionBirAbifunctional ligase/repressorBCCPbiotin-carboxylase cargo proteinBPLbiotin protein ligaseEMARSenzyme-mediated activation of radical sourcesERendoplasmic reticulumIDRintrinsically disordered regionivBioIDin vitro BioIDFKBPFK506-binding proteinFRBFKBP-rapamycin binding domainHRPhorseradish peroxidase CNHS-biotinN-hydroxysuccinimide-biotinPCAprotein-fragment complementation assayPDB-MSproximity-dependent biotinylation coupled to mass spectrometrySPPLATselective proteomic proximity labeling using tyramideSILACstable isotope labeling by amino acids in cell cultureTMTtandem mass tagiTRAQisobaric tag for relative and absolute quantification. approaches and consist of directing an enzyme capable of catalyzing covalent transfer of biotin (or other derivatives) to endogenous proteins that are located within a certain distance of the enzyme. By fusing the enzyme to specific proteins (referred to as "baits"), the enzyme can be localized to distinct areas of the cell, for example to a protein complex or an organelle (Fig. 1A). Addition of the enzyme substrate leads to the covalent biotinylation of proteins located near the bait (these are referred to as "preys"). Importantly, the labeling can be performed in live cells (or whole organisms), on fixed samples, or even in lysates or semi-purified structures. The primary advantage of PDB is that protein-protein interactions or the integrity of organelles do not need to be maintained post-labeling as the covalently biotinylated preys can be captured using an affinity matrix, most often streptavidin. This principle has enabled purification of preys under harsh lysis and wash conditions because of the high affinity of the biotin-streptavidin interaction (Kd ∼10−14m), which is also resistant to many conditions (detergents, salt, or denaturing agents) that typically disrupt protein-protein interactions or organellar integrity. Subsequently, streptavidin-purified proteins can be identified and quantified by mass spectrometry (Fig. 1B). When appropriate controls and mass spectrometric quantification are employed (as discussed elsewhere - (18Trinkle-Mulcahy L. Recent advances in proximity-based labeling methods for interactome mapping.F1000Research. 2019; 8Crossref PubMed Scopus (18) Google Scholar, 19Gingras A.C. Abe K.T. Raught B. Getting to know the neighborhood: using proximity-dependent biotinylation to characterize protein complexes and map organelles.Curr. Opinion Chem. Biol. 2019; 48: 44-54Crossref PubMed Scopus (0) Google Scholar, 20Varnaite R. MacNeill S.A. Meet the neighbors: Mapping local protein interactomes by proximity-dependent labeling with BioID.Proteomics. 2016; 16: 2503-2518Crossref PubMed Scopus (59) Google Scholar)), 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. Additionally, these PDB-MS approaches will reveal the proximal relationships of a bait throughout the entire life cycle of the protein from its synthesis until the end point of the assay. This is discussed below (in Proximal Labeling Chemistry and Labeling Propensity) and elsewhere (19Gingras A.C. Abe K.T. Raught B. Getting to know the neighborhood: using proximity-dependent biotinylation to characterize protein complexes and map organelles.Curr. Opinion Chem. Biol. 2019; 48: 44-54Crossref PubMed Scopus (0) Google Scholar). proximity-dependent biotinylation affinity purification coupled to mass spectrometry ascorbate peroxidase biotinylation by antibody recognition bifunctional ligase/repressor biotin-carboxylase cargo protein biotin protein ligase enzyme-mediated activation of radical sources endoplasmic reticulum intrinsically disordered region in vitro BioID FK506-binding protein FKBP-rapamycin binding domain horseradish peroxidase C N-hydroxysuccinimide-biotin protein-fragment complementation assay proximity-dependent biotinylation coupled to mass spectrometry selective proteomic proximity labeling using tyramide stable isotope labeling by amino acids in cell culture tandem mass tag isobaric tag for relative and absolute quantification. proximity-dependent biotinylation affinity purification coupled to mass spectrometry ascorbate peroxidase biotinylation by antibody recognition bifunctional ligase/repressor biotin-carboxylase cargo protein biotin protein ligase enzyme-mediated activation of radical sources endoplasmic reticulum intrinsically disordered region in vitro BioID FK506-binding protein FKBP-rapamycin binding domain horseradish peroxidase C N-hydroxysuccinimide-biotin protein-fragment complementation assay proximity-dependent biotinylation coupled to mass spectrometry selective proteomic proximity labeling using tyramide stable isotope labeling by amino acids in cell culture tandem mass tag isobaric tag for relative and absolute quantification. Since the introduction of the first PDB-MS approach (reviewed in (18Trinkle-Mulcahy L. Recent advances in proximity-based labeling methods for interactome mapping.F1000Research. 2019; 8Crossref PubMed Scopus (18) Google Scholar, 19Gingras A.C. Abe K.T. Raught B. Getting to know the neighborhood: using proximity-dependent biotinylation to characterize protein complexes and map organelles.Curr. Opinion Chem. Biol. 2019; 48: 44-54Crossref PubMed Scopus (0) Google Scholar, 21Rees J.S. Li X.W. Perrett S. Lilley K.S. Jackson A.P. Protein Neighbors and Proximity Proteomics.Mol. Cell. Proteomics. 2015; 14: 2848-2856Abstract Full Text Full Text PDF PubMed Scopus (59) Google Scholar, 22Kim D.I. Roux K.J. Filling the void: proximity-based labeling of proteins in living cells.Trends Cell Biol. 2016; 26: 804-817Abstract Full Text Full Text PDF PubMed Scopus (106) Google Scholar)), 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 proximity-dependent biotinylation approaches and on the development of distinct toolsets for the application of proximity dependent biotinylation to different biological questions. In this section, we will describe the two major classes of enzymes currently used for PDB-MS: biotin protein ligases and peroxidases, with an emphasis on their structural characteristics, their natural enzymatic reactions, and the modifications that have made BioID and APEX possible. Biotin is an essential vitamin for all organisms that is produced by plants, fungi and most prokaryotes, but not mammalian cells (23Tong L. Structure and function of biotin-dependent carboxylases.Cell Mol. Life Sci. 2013; 70: 863-891Crossref PubMed Scopus (162) Google Scholar). In mammalian cells, biotin uptake is primarily mediated by the sodium multivitamin transporter SMVT (encoded by the SLC5A6 gene in humans) (24Azhar A. Booker G. Polyak S. Mechanisms of biotin transport.Biochem. Anal. Biochem. 2015; 4 (1000210-1000211-1000218)Google Scholar). Intracellular biotin serves as a covalently-attached cofactor for the biotin-dependent carboxylase enzymes (four enzyme families are present in humans: PC, PCCA/PCCB, MCCC1/MCCC2, ACACA/ACACB) that have crucial roles in amino acid, fatty acid, carbohydrate and energy metabolism. Carboxylases transfer carboxyl groups to small molecule substrates, most of which are coenzyme A (CoA) esters of organic acids (though other compounds including urea and pyruvate can also serve as substrates). This transfer is enabled by biotin, which first becomes enzymatically carboxylated (bicarbonate serves as the CO2 donor), and in a second enzymatic step, releases the carboxyl group to the substrate (for a review of carboxylases and their mechanisms, please refer to (23Tong L. Structure and function of biotin-dependent carboxylases.Cell Mol. Life Sci. 2013; 70: 863-891Crossref PubMed Scopus (162) Google Scholar)). Biotin protein ligases (here referred to as BPLs), also known as holocarboxylase synthetases in eukaryotes, are responsible for the covalent attachment of biotin to the carboxylases (25Chapman-Smith A. Cronan Jr., J.E. Molecular biology of biotin attachment to proteins.J. Nutrition. 1999; 129: 477S-484SCrossref PubMed Google Scholar), and are present in all living species. They exhibit a high substrate specificity for the carboxylases and this has been evolutionarily conserved as specific biotinylation can still occur when the BPL and carboxylase come from divergent species (26McAllister H.C. Coon M.J. Further studies on the properties of liver propionyl coenzyme A holocarboxylase synthetase and the specificity of holocarboxylase formation.J. Biol. Chem. 1966; 241: 2855-2861Abstract Full Text PDF PubMed Google Scholar). This high specificity for a very small number of substrates (largely localized in the mitochondrial matrix in eukaryotes) is important for the use of BPLs in several biotechnology applications, including BioID. BPL enzymes (PFAM: PF03099) can be grouped into three classes based on their structural architecture (27Sternicki L.M. Wegener K.L. Bruning J.B. Booker G.W. Polyak S.W. Mechanisms Governing Precise Protein Biotinylation.Trends biochemical sciences. 2017; 42: 383-394Abstract Full Text Full Text PDF PubMed Scopus (0) Google Scholar) (Fig. 2A). All three classes have a conserved central catalytic domain responsible for the protein biotinylation reaction and a C-terminal domain of unknown function that is essential for its enzymatic activity (28Chapman-Smith A. Mulhern T.D. Whelan F. Cronan Jr, J.E. Wallace J.C. The C-terminal domain of biotin protein ligase from E. coli is required for catalytic activity.Protein Sci. 2001; 10: 2608-2617Crossref PubMed Scopus (54) Google Scholar). However, they differ in their N termini. Class II enzymes possess a winged helix-turn-helix DNA-binding domain that functions as a biotin-controlled repressor of the biotin biosynthesis operon (29Xu Y. Beckett D. Evidence for interdomain interaction in the Escherichia coli repressor of biotin biosynthesis from studies of an N-terminal domain deletion mutant.Biochemistry. 1996; 35: 1783-1792Crossref PubMed Scopus (37) Google Scholar), whereas class III enzymes have a large N terminus without DNA binding activity. Class I enzymes lack this N-terminal domain altogether. Class I and II BPLs are found in Archaea, prokaryotes and plants whereas class III BPLs are found in yeast, insects and mammals and include the human holocarboxylase synthetase HLCS (Fig. 2A). As discussed below, class II BPLs (such as that found in E. coli) have been extensively used in PDB-MS and in biotechnology in general, whereas class I enzymes (such as that found in Aquifex aeolicus, a thermophilic bacteria) have been more recently introduced for use in PDB-MS and have unique properties (30Kim D.I. Jensen S.C. Noble K.A. Kc B. Roux K.H. Motamedchaboki K. Roux K.J. An improved smaller biotin ligase for BioID proximity labeling.Mol. Biol. Cell. 2016; 27: 1188-1196Crossref PubMed Scopus (200) Google Scholar). The structure and activity of several BPLs from different bacterial species have been described, providing insight into their reaction mechanism (27Sternicki L.M. Wegener K.L. Bruning J.B. Booker G.W. Polyak S.W. Mechanisms Governing Precise Protein Biotinylation.Trends biochemical sciences. 2017; 42: 383-394Abstract Full Text Full Text PDF PubMed Scopus (0) Google Scholar, 31Feng J. Paparella A.S. Booker G.W. Polyak S.W. Abell A.D. Biotin protein ligase is a target for new antibacterials.Antibiotics. 2016; 5 (pii): E26Crossref Scopus (11) Google Scholar). The E. coli BPL, also known as Bifunctional ligase/repressor (BirA), is an archetypal type-II enzyme and is one of the best-studied enzymes of this class. Upon binding of biotin to BirA, the biotin-binding loop undergoes a conformational change that allows for subsequent binding of ATP, leading to a structural rearrangement of the adenylate-binding loop, stabilizing the bound ATP (Fig. 3). Subsequently, a nucleophilic substitution mediated by K183 of BirA catalyzes the attack of the biotin carboxylate on the alpha phosphate of ATP, producing biotinyl-5′-AMP. Biotinyl-5′-AMP remains stably associated with the enzyme in a mixed anhydride form through hydrogen bonding with the R118 backbone (27Sternicki L.M. Wegener K.L. Bruning J.B. Booker G.W. Polyak S.W. Mechanisms Governing Precise Protein Biotinylation.Trends biochemical sciences. 2017; 42: 383-394Abstract Full Text Full Text PDF PubMed Scopus (0) Google Scholar, 32Weaver L.H. Kwon K. Beckett D. Matthews B.W. Corepressor-induced organization and assembly of the biotin repressor: a model for allosteric activation of a transcriptional regulator.Proc. Natl. Acad. Sci. 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The second step of the transfer reaction involves the nucleophilic attack of this mixed anhydride by the epsilon amine of lysine from the substrate (K122 on the biotin-carboxylase cargo protein, BCCP, a subunit of the acetyl-CoA carboxylase), resulting in covalent biotinylation of BCCP on the attacking lysine (25Chapman-Smith A. Cronan Jr., J.E. Molecular biology of biotin attachment to proteins.J. Nutrition. 1999; 129: 477S-484SCrossref PubMed Google Scholar, 34Yao X. Wei D. Soden Jr, C. Summers M.F. Beckett D. Structure of the carboxy-terminal fragment of the apo-biotin carboxyl carrier subunit of Escherichia coli acetyl-CoA carboxylase.Biochemistry. 1997; 36: 15089-15100Crossref PubMed Scopus (65) Google Scholar, 35Streaker E.D. Beckett D. Nonenzymatic biotinylation of a biotin carboxyl carrier protein: unusual reactivity of the physiological target lysine.Protein Sci. 2006; 15: 1928-1935Crossref PubMed Scopus (0) Google Scholar). 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Biotinylation of proteins in vivo: a useful posttranslational modification for protein analysis.Methods Enzymol. 2000; 326: 440-458Crossref PubMed Google Scholar), or protein visualization using fluorophores conjugated to streptavidin (43Chen I. Howarth M. Lin W. Ting A.Y. Site-specific labeling of cell surface proteins with biophysical probes using biotin ligase.Nat. Methods. 2005; 2: 99-104Crossref PubMed Scopus (497) Google Scholar). Other applications include the tagging of ribosomes localized to different parts of the cell to elucidate which transcripts they translate (44Jan C.H. Williams C.C. Weissman J.S. Principles of ER cotranslational translocation revealed by proximity-specific ribosome profiling.Science. 2014; 346: 1257521Crossref PubMed Scopus (168) Google Scholar), and the selective purification of structures, e.g. the nucleus, to assist in downstream assay design (e.g. (45Deal R.B. Henikoff S. The INTACT method for cell type-specific gene expression and chromatin profiling in Arabidopsis thaliana.Nat. Protoc. 2011; 6: 56-68Crossref PubMed Scopus (235) Google Scholar)). Importantly, however, this application of BirA requires the expression of two proteins, one fused to BirA and one fused to the AviTag, which limits discovery proteomics assays. Although the wildtype BirA remains widely used, many BirA mutants have been described over the years, presenting opportunities for new applications (Fig. 4). The study of mutants that affect the biotin operon activity in E. coli resulted in identification of the BirA91 mutant allele in 1980 (46Barker D.F. Campbell A.M. Use of bio-lac fusion strains to study regulation of biotin biosynthesis in Escherichia coli.J. Bacteriol. 1980; 143: 789-800Crossref PubMed Google Scholar), with the specific mutation (R118G) identified in 1986 (47Buoncristiani M.R. Howard P.K. Otsuka A.J. DNA-binding and enzymatic domains of the bifunctional biotin operon repressor (BirA) of Escherichia coli.Gene. 1986; 44: 255-261Crossref PubMed Google Scholar). Relative to wildtype BirA, this mutant was found to have 100-fold greater Kd for biotin and a 400-fold higher dissociation rate for biotinyl-5′-AMP (48Kwon K. Beckett D. Function of a conserved sequence motif in biotin holoenzyme synthetases.Protein Sci. 2000; 9: 1530-1539Crossref PubMed Google Scholar, 49Kwon K. Streaker E.D. Ruparelia S. Beckett D. Multiple disordered loops function in corepressor-induced dimerization of the biotin repressor.J. Mol. Biol. 2000; 304: 821-833Crossref PubMed Scopus (46) Google Scholar, 50Cronan J.E. Targeted and proximity-dependent promiscuous protein biotinylation by a mutant Escherichia coli biotin protein ligase.J. Nutritional Biochem. 2005; 16: 416-418Crossref PubMed Scopus (0) Google Scholar), consistent with the role of R118 in stabilizing the biotinyl-5′-AMP intermediate. Later, the R118G mutant was demonstrated to act as a non-sequence specific biotinylation reagent by Choi-Rhee et al., who first described the potential use of this mutant for the "recovery of interacting proteins by existing avidin/streptavidin technology" (51Choi-Rhee E. Schulman H. Cronan J.E. Promiscuous protein biotinylation by Escherichia coli biotin protein ligase.Protein Sci. 2004; 13: 3043-3050Cr
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