Deciphering molecular interactions by proximity labeling.

Proximity labeling techniques are emerging high-throughput methods for studying protein-protein, protein-RNA, and protein-DNA interactions with temporal and spatial precision. Proximity labeling methods take advantage of enzymes that can covalently label biomolecules with reactive substrates. These labeled biomolecules can be identified using mass spectrometry or next-generation sequencing. The main advantage of these methods is their ability to capture weak or transient interactions between biomolecules. Proximity labeling is indispensable for studying organelle interactomes. Additionally, it can be used to resolve spatial composition of macromolecular complexes. Many of these methods have only recently been introduced; nonetheless, they have already provided new and deep insights into the biological processes at the cellular, organ, and organism levels. In this paper, we review a broad range of proximity labeling techniques, their development, drawbacks and advantages, and implementations in recent studies.

Protein‒protein, protein‒RNA, and protein‒DNA interaction networks form the basis of cellular regulation and signal transduction, making it crucial to explore these interaction networks to understand complex biological processes. Traditional methods such as affinity purification and yeast two-hybrid assays have been shown to have limitations, as they can only isolate high-affinity molecular interactions under nonphysiological conditions or in vitro. Moreover, these methods have shortcomings for organelle isolation and protein subcellular localization. To address these issues, proximity labeling techniques have been developed. This technology not only overcomes the limitations of traditional methods but also offers unique advantages in studying protein spatial characteristics and molecular interactions within living cells. Currently, this technique not only is indispensable in research on mammalian nucleoprotein interactions but also provides a reliable approach for studying nonmammalian cells, such as plants, parasites and viruses. Given these advantages, this article provides a detailed introduction to the principles of proximity labeling techniques and the development of labeling enzymes. The focus is on summarizing the recent applications of TurboID and miniTurbo in mammals, plants, and microorganisms.D-8DQMfpJtz9TZZfzFbSsiVideo

Preface

Furan mediated nucleic acid cross-linking, initially developed for DNA interstrand duplex cross-linking, has matured into a versatile tool for the study of protein and nucleic acid interactions, ready to face its applications. The methodology was initially developed for easy and clean chemical generation of DNA interstrand cross-linked duplexes, but has been further expanded for use with other probes, targets and triggers, now allowing mild biologically significant cross-linking with potential therapeutic benefit. It was shown that the methodology could be repurposed for RNA interstrand cross-linking, which is very relevant in today's antisense approaches or miRNA target identification endeavors. This further illustrates the furan oxidation method's generality and mildness, especially when using red light for oxidation. A complementary antigene approach has been explored through duplex targeting with furan modified triplex forming oligonucleotides (TFOs) and DNA binding proteins. Also targeting of peptides and proteins by furan-modified DNA and peptides has been explored. Thorough methodology examination exploring variable reaction conditions in combination with a series of different furan-modified building blocks and application of different activation signals resulted in a detailed understanding of the mechanisms involved and factors influencing the yield and selectivity of the reaction. In order to draw the bigger picture of the scope and limitations of furan-oxidation cross-linking, we here provide a unique side by side comparison and discussion of our published data, supplemented with unpublished results, providing a clear performance report of the currently established furan toolbox and its application potential in various biomacromolecular complexes.

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The Ku heterodimer plays an essential role in non-homologous end-joining and other cellular processes including transcription, telomere maintenance and apoptosis. While the function of Ku is regulated through its association with other proteins and nucleic acids, the specific composition of these macromolecular complexes and their dynamic response to endogenous and exogenous cellular stimuli are not well understood. Here we use quantitative proteomics to define the composition of Ku multicomponent complexes and demonstrate that they are dramatically altered in response to UV radiation. Subsequent biochemical assays revealed that the presence of DNA ends leads to the substitution of RNA-binding proteins with DNA and chromatin associated factors to create a macromolecular complex poised for DNA repair. We observed that dynamic remodeling of the Ku complex coincided with exit of Ku and other DNA repair proteins from the nucleolus. Microinjection of sheared DNA into live cells as a mimetic for double strand breaks confirmed these findings in vivo.

Proximity labeling: an emerging tool for probing in planta molecular interactions

Simple SummaryT-cell intracellular antigen 1 (TIA1) is a DNA/RNA-binding protein best known for its different roles in RNA metabolism. Currently, little is known about the interacting protein partners of TIA1 in control and stress conditions that could shed light on its multiple context-specific molecular functions. Proximity labeling is a technique in which a labeling enzyme, here APEX2, that is fused to the protein of interest, in this case TIA1, marks the protein’s interacting network in living cells, allowing for subsequent ex vivo analysis using protein identification methods such as mass spectrometry. Hereby, combining these two techniques, it was revealed that the TIA1 interactome has very distinct protein partners in control and unstressed cells and that these partners are involved in not only previously identified processes such as splicing, nucleocytoplasmic transport, and different levels of protein translation control, but also in novel ones such as DNA double-strand break repair and mitochondrial metabolism. Overall, these findings provide a more precise definition of TIA1’s function in cells and pave the way to dissect its role in each of these processes.TIA1 is a broadly expressed DNA/RNA binding protein that regulates multiple aspects of RNA metabolism. It is best known for its role in stress granule assembly during the cellular stress response. Three RNA recognition motifs mediate TIA1 functions along with a prion-like domain that supports multivalent protein-protein interactions that are yet poorly characterized. Here, by fusing the enhanced ascorbate peroxidase 2 (APEX2) biotin-labeling enzyme to TIA1 combined with mass spectrometry, the proteins in the immediate vicinity of TIA1 were defined in situ. Eighty-six and 203 protein partners, mostly associated with ribonucleoprotein complexes, were identified in unstressed control and acute stress conditions, respectively. Remarkably, the repertoire of TIA1 protein partners was highly dissimilar between the two cellular states. Under unstressed control conditions, the biological processes associated with the TIA1 interactome were enriched for cytosolic ontologies related to mRNA metabolism, such as translation initiation, nucleocytoplasmic transport, and RNA catabolism, while the protein identities were primarily represented by RNA binding proteins, ribosomal subunits, and eicosanoid regulators. Under acute stress, TIA1-labeled partners displayed a broader subcellular distribution that included the chromosomes and mitochondria. The enriched biological processes included splicing, translation, and protein synthesis regulation, while the molecular function of the proteins was enriched for RNA binding activity, ribosomal subunits, DNA double-strand break repair, and amide metabolism. Altogether, these data highlight the TIA1 spatial environment with its different partners in diverse cellular states and pave the way to dissect TIA1 role in these processes.

High throughput genomic/proteomic strategies, such as microarray studies, drug screens, and genetic screens, often produce a list of genes that are believed to be important for one or more reasons. Unfortunately it is often difficult to discern meaningful biological relationships from such lists. This study presents a new bioinformatic approach that can be used to identify regulatory subnetworks for lists of significant genes or proteins. We demonstrate the utility of this approach using an interaction network for yeast constructed from BIND, TRANSFAC, SCPD, and chromatin immunoprecipitation (ChIP)-Chip data bases and lists of genes from well known metabolic pathways or differential expression experiments. The approach accurately rediscovers known regulatory elements of the heat shock response as well as the gluconeogenesis, galactose, glycolysis, and glucose fermentation pathways in yeast. We also find evidence supporting a previous conjecture that approximately half of the enzymes in a metabolic pathway are transcriptionally co-regulated. Finally we demonstrate a previously unknown connection between GAL80 and the diauxic shift in yeast.

Growing numbers of protein and nucleic acid complex structures are being determined and deposited in the Protein Data Bank and the Nucleic Acid Database. With the increasing complexity of these structures, it is challenging to analyse and visualize the three-dimensional interactions. The currently available programs for such analysis and visualization are limited in their applications. They can only analyse a subset of protein-nucleic acid complexes and require multiple iterations before obtaining plots that are suitable for presentation. An interactive web-based program, NuProPlot (http://www.nuproplot.com), has been developed which can automatically identify hydrogen, electrostatic and van der Waals interactions between proteins and nucleic acids and generate a plot showing all of the interactions. Protein-DNA and protein-RNA interactions can be visualized in simple two-dimensional schematics. Interactive schematic drawing options allow selection of the plotted area and repositioning of the individual interactions for better legibility. NuProPlot is a fully automated and user-friendly program providing various custom options. NuProPlot represents a greatly improved option for analysis and presentation of protein-nucleic acid interactions.

Multisite phosphorylation of proteins is a general mechanism for modulation of protein function and molecular interactions. Definition of phosphorylation sites and elucidation of the functional interplay between multiple phosphorylated residues in proteins are, however, a major analytical challenge in current molecular cell biology and proteomic research. In the present study, we used mass spectrometry to determine the major phosphorylated residues of the human epidermal growth factor (EGF) receptor at various well defined cellular conditions. Activation of EGF receptor was achieved by several types of stimulation, i.e. by sodium pervanadate, EGF, and integrin-dependent adhesion. The contribution of cell-matrix adhesion was also determined by activating the EGF receptor by EGF in cells kept in suspension. We developed an analytical strategy that combined miniaturized sample preparation techniques and MALDI tandem mass spectrometry and determined a total of nine phosphorylation sites in the EGF receptor. We discovered one novel phosphorylation site (Ser967) and revealed constitutive phosphorylation of Thr669, Ser967, Ser1002, and Tyr1045 and stimulation-dependent differential phosphorylation of Tyr1068, Tyr1086, Ser1142, Tyr1148, and Tyr1173. The EGF receptor was purified from HeLa cells or ECV304 cells by immunoprecipitation and SDS-PAGE and then digested with trypsin. Phosphopeptides in the range of 0.8-3.7 kDa were recovered by combinations of IMAC, perfusion chromatography, and graphite powder chromatography and subsequently detected and sequenced by MALDI quadrupole time-of-flight tandem mass spectrometry. Two phosphorylation sites were detected in the peptide 1137GSHQISLDNPDYQQDFFPK1155; however, only Tyr1148 was phosphorylated upon EGF treatment; in contrast Ser1142 was only phosphorylated by integrin-dependent adhesion in the absence of EGF treatment, suggesting differential phosphorylation of this region by distinct stimuli. This MALDI MS/MS-based analytical approach demonstrates the feasibility of systematic analysis of signaling molecules by mass spectrometry and provides new insights into the dynamics of receptor signaling processes.

ConspectusProximitylabeling can be defined as an enzymatic “in-cell”chemical reaction that catalyzes the proximity-dependent modificationof biomolecules in live cells. Since the modified proteins can beisolated and identified via mass spectrometry, this method has beensuccessfully utilized for the characterization of local proteomessuch as the sub-mitochondrial proteome and the proteome at membranecontact sites, or spatiotemporal interactome information in live cells,which are not “accessible” via conventional methods.Currently, proximity labeling techniques can be applied not only forlocal proteome mapping but also for profiling local RNA and DNA, inaddition to showing great potential for elucidating spatial cell–cellinteraction networks in live animal models. We believe that proximitylabeling has emerged as an essential tool in “spatiomics,”that is, for the extraction of spatially distributed biological informationin a cell or organism.Proximity labeling is a multidisciplinarychemical technique. Fora decade, we and other groups have engineered it for multiple applicationsbased on the modulation of enzyme chemistry, chemical probe design,and mass analysis techniques that enable superior mapping results.The technique has been adopted in biology and chemistry. This “in-cell”reaction has been widely adopted by biologists who modified it intoan in vivo reaction in animal models. In our laboratory, we conductedin vivo proximity labeling reactions in mouse models and could successfullyobtain the liver-specific secretome and muscle-specific mitochondrialmatrix proteome. We expect that proximity reaction can further contributeto revealing tissue-specific localized molecular information in liveanimal models.Simultaneously, chemists have also adopted theconcept and employedchemical “photocatalysts” as artificial enzymes to developnew proximity labeling reactions. Under light activation, photocatalystscan convert the precursor molecules to the reactive species via electrontransfer or energy transfer and the reactive molecules can react withproximal biomolecules within a definite lifetime in an aqueous solution.To identify the modified biomolecules by proximity labeling, the modifiedbiomolecules should be enriched after lysis and sequenced using sequencingtools. In this analysis step, the direct detection of modified residue(s)on the modified proteins or nucleic acids can be the proof of theirlabeling event by proximal enzymes or catalysts in the cell. In thisAccount, we introduce the basic concept of proximity labeling andthe multidirectional advances in the development of this method. Webelieve that this Account may facilitate further utilization and modificationof the method in both biological and chemical research communities,thereby revealing unknown spatially distributed molecular or cellularinformation or spatiome.

A Protein Data Bank study was conducted to check the role of C-halogen(X)…pi interactions (X = F, Cl, Br, I) in nucleic acids. The presence of halogens can be attributed to not only the modified residues of the nucleic acid but also the ones associated with the ligand. The study reports the presence of bromine amongst modified residues of the nucleic acid being the maximum than any other halogens. It is important to consider these interactions as they seem to be responsible for increasing the affinity of the ligand to the biomolecule concerned. The role of halogen-pi interactions becomes important to be assessed in order to design halogenated ligands that will certainly be more effective in increasing the binding of the corresponding ligand to the target nucleic acid. The study attempts at listing the halogens that are prominent in the existing nucleic acid and ligand interactions which are deposited in the PDB. Inter-atomic interactions based on C-X…pi were analyzed to be found in several PDB IDs and similar ligand halogenation can be carried out to effectively increase binding affinity of halogenated ligands to nucleic acid based targets in case of several diseases. SYNOPSIS A comprehensive analysis of C-halogen…pi interaction (C-X…pi) among available nucleic acid structures in RCSB PDB has been presented pointing to their application in ligand modification to improve interaction with biomolecules. Existing structures point to the presence of these non-bonded interactions in several diseases.

Extracellular proteins play pivotal roles in both intracellular signaling and intercellular communications in health and disease. While recent advancements in proximity labeling (PL) methods, such as peroxidase- and photocatalyst-based approaches, have facilitated the resolution of extracellular proteomes, their in vivo compatibility remains limited. Here, we report TyroID, an in vivo-compatible PL method for unbiased mapping of extracellular proteins with high spatiotemporal resolution. TyroID employs plant- and bacteria-derived tyrosinases to produce reactive o-quinone intermediates, enabling the labeling of multiple residues on endogenous proteins with bioorthogonal handles, thereby allowing for their identification via chemical proteomics. We validate TyroID's specificity by mapping extracellular proteomes and HER2-neighboring proteins using nanobody-directed recombinant tyrosinases. Demonstrating its superiority over other PL methods, TyroID enables in vivo mapping of extracellular proteomes, including mapping HER2-proximal proteins in tumor xenografts, quantifying the turnover of plasma proteins and labeling hippocampal-specific proteomes in live mouse brains. TyroID emerges as a potent tool for investigating protein localization and molecular interactions within living organisms.

Extracellular proteins play pivotal roles in both intracellular signaling and intercellular communications in health and disease. While recent advancements in proximity labeling (PL) methods, such as peroxidase- and photocatalyst-based approaches, have facilitated the resolution of extracellular proteomes, their in vivo compatibility remains limited. Here, we report TyroID, an in vivo-compatible PL method for the unbiased mapping of extracellular proteins with high spatiotemporal resolution. TyroID employs plant- and bacteria-derived tyrosinases to produce reactive o-quinone intermediates, enabling the labeling of multiple residues on endogenous proteins with bioorthogonal handles, thereby allowing for their identification via chemical proteomics. We validate TyroID’s specificity by mapping extracellular proteomes and HER2-neighboring proteins using affibody-directed recombinant tyrosinases. Demonstrating its superiority over other PL methods, TyroID enables in vivo mapping of extracellular proteomes, including mapping HER2-proximal proteins in tumor xenografts, quantifying the turnover of plasma proteins and labeling hippocampal-specific proteomes in live mouse brains. TyroID emerges as a potent tool for investigating protein localization and molecular interactions within living organisms.