Proximity Labeling
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Enzyme-catalyzed proximity labeling (PL), also known as proximity-based labeling, is a
laboratory technique A laboratory (; ; colloquially lab) is a facility that provides controlled conditions in which scientific or technological research, experiments, and measurement may be performed. Laboratory services are provided in a variety of settings: physicia ...
that labels
biomolecule A biomolecule or biological molecule is a loosely used term for molecules present in organisms that are essential to one or more typically biological processes, such as cell division, morphogenesis, or development. Biomolecules include large ...
s, usually proteins or RNA, proximal to a protein of interest. By creating a
gene fusion A fusion gene is a hybrid gene formed from two previously independent genes. It can occur as a result of translocation, interstitial deletion, or chromosomal inversion. Fusion genes have been found to be prevalent in all main types of human neoplas ...
in a living cell between the protein of interest and an engineered labeling enzyme, biomolecules spatially proximal to the protein of interest can then be selectively marked with
biotin Biotin (or vitamin B7) is one of the B vitamins. It is involved in a wide range of metabolic processes, both in humans and in other organisms, primarily related to the utilization of fats, carbohydrates, and amino acids. The name ''biotin'', bor ...
for pulldown and analysis. Proximity labeling has been used for identifying the components of novel cellular structures and for determining protein-protein interaction partners, among other applications.


History

Before the development of proximity labeling, determination of protein proximity in cells relied on studying protein-protein interactions through methods such as affinity purification-mass spectrometry and proximity ligation assays. DamID is a method developed in 2000 by
Steven Henikoff Steven Henikoff is a scientist at the Fred Hutchinson Cancer Research Center, and an HHMI Investigator. His field of study is chromatin-related transcriptional regulation. He earned his BS in chemistry at the University of Chicago. He earned his P ...
for identifying parts of the genome proximal to a chromatin protein of interest. DamID relies on a DNA methyltransferase fusion to the chromatin protein to nonnaturally methylate DNA, which can then be subsequently sequenced to reveal genome methylation sites near the protein. Researchers were guided by the fusion protein strategy of DamID to create a method for site-specific labeling of protein targets, culminating in the creation of the biotin protein labelling-based BioID in 2012.
Alice Ting Alice Yen-Ping Ting is Taiwanese-born American chemist. She is a professor of Genetics, of Biology, and by courtesy, of Chemistry at Stanford University. She is also a Chan Zuckerberg Biohub investigator. Early life and education Alice Ting w ...
and the Ting lab at
Stanford University Stanford University, officially Leland Stanford Junior University, is a private research university in Stanford, California. The campus occupies , among the largest in the United States, and enrolls over 17,000 students. Stanford is consider ...
have engineered several proteins that demonstrate improvements in biotin-based proximity labeling efficacy and speed.


Principles

Proximity labeling relies on a labeling enzyme that can biotinylate nearby biomolecules promiscuously. Biotin labeling can be achieved through several different methods, depending on the species of labeling enzyme. * BioID, also known as BirA*, is a mutant ''E. coli'' biotin ligase that catalyzes the activation of biotin by ATP. The activated biotin is short-lived and thus can only diffuse to a region proximal to BioID. Labeling is achieved when the activated biotin reacts with nearby amines, such as the
lysine Lysine (symbol Lys or K) is an α-amino acid that is a precursor to many proteins. It contains an α-amino group (which is in the protonated form under biological conditions), an α-carboxylic acid group (which is in the deprotonated −C ...
sidechain amines found in proteins. TurboID is a biotin ligase engineered via yeast surface display directed evolution. TurboID enables ~10 minute labeling times instead of the ~18 hour labeling times required by BioID. * APEX is an ascorbate peroxidase derivative reliant on hydrogen peroxide for catalyzing the oxidation of biotin-tyramide, also known as biotin-phenol, to a short-lived and reactive biotin-phenol free radical. Labeling is achieved when this intermediate reacts with various functional groups of nearby biomolecules. APEX can also be used for local deposition of diaminobenzidine, a precursor for an electron microscopy stain. APEX2 is a derivative of APEX engineered via yeast surface display directed evolution. APEX2 shows improved labeling efficiency and cellular expression levels. To label proteins nearby a protein of interest, a typical proximity labeling experiment begins by cellular expression of an APEX2 fusion to the protein of interest, which localizes to the protein of interest's native environment. Cells are next incubated with biotin-phenol, then briefly with hydrogen peroxide, initiating biotin-phenol free radical generation and labeling. To minimize cellular damage, the reaction is then quenched using an antioxidant buffer. Cells are lysed and the labeled proteins are pulled down with
streptavidin Streptavidin is a 66.0 (tetramer) kDa protein purified from the bacterium '' Streptomyces avidinii''. Streptavidin homo-tetramers have an extraordinarily high affinity for biotin (also known as vitamin B7 or vitamin H). With a dissociation co ...
beads. The proteins are digested with trypsin, and finally the resulting peptidic fragments are analyzed using shotgun proteomics methods such as LC-MS/MS or SPS-MS3. If instead a protein fusion is not genetically accessible (such as in human tissue samples) but an antibody for the protein of interest is known, proximity labeling can still be enabled by fusing a labeling enzyme with the antibody, then incubating the fusion with the sample.


Applications

Proximity labeling methods have been used to study the proteomes of biological structures that are otherwise difficult to isolate purely and completely, such as
cilia The cilium, plural cilia (), is a membrane-bound organelle found on most types of eukaryotic cell, and certain microorganisms known as ciliates. Cilia are absent in bacteria and archaea. The cilium has the shape of a slender threadlike projecti ...
,
mitochondria A mitochondrion (; ) is an organelle found in the Cell (biology), cells of most Eukaryotes, such as animals, plants and Fungus, fungi. Mitochondria have a double lipid bilayer, membrane structure and use aerobic respiration to generate adenosi ...
, postsynaptic clefts, p-bodies,
stress granule Stress granules are dense aggregations in the cytosol composed of proteins and RNAs that appear when the cell is under stress. The RNA molecules stored are stalled translation pre-initiation complexes: failed attempts to make protein from mRNA. St ...
s, and lipid droplets. Fusion of APEX2 with G-protein coupled receptors (GPCRs) allows for both tracking GPCR signaling at a 20 second temporal resolution and also identification of unknown GPCR-linked proteins. Proximity labeling has also been used for transcriptomics and interactomics. In 2019,
Alice Ting Alice Yen-Ping Ting is Taiwanese-born American chemist. She is a professor of Genetics, of Biology, and by courtesy, of Chemistry at Stanford University. She is also a Chan Zuckerberg Biohub investigator. Early life and education Alice Ting w ...
and the Ting lab have used APEX to identify RNA localized to specific cellular compartments. In 2019, BioID has been tethered to the beta-actin mRNA transcript to study its localization dynamics. Proximity labeling has also been used to find interaction partners of heterodimeric
protein phosphatases A protein phosphatase is a phosphatase enzyme that removes a phosphate group from the phosphorylated amino acid residue of its substrate protein. Protein phosphorylation is one of the most common forms of reversible protein posttranslational modif ...
, of the miRISC (microRNA-induced silencing complex) protein Ago2, and of
ribonucleoproteins Nucleoproteins are proteins conjugated with nucleic acids (either DNA or RNA). Typical nucleoproteins include ribosomes, nucleosomes and viral nucleocapsid proteins. Structures Nucleoproteins tend to be positively charged, facilitating inte ...
.


Recent developments

TurboID-based proximity labeling has been used to identify regulators of a receptor involved in the
innate immune response The innate, or nonspecific, immune system is one of the two main immunity strategies (the other being the adaptive immune system) in vertebrates. The innate immune system is an older evolutionary defense strategy, relatively speaking, and is the ...
, a NOD-like receptor. BioID-based proximity labeling has been used to identify the molecular composition of breast cancer cell
invadopodia Invadopodia are actin-rich protrusions of the plasma membrane that are associated with degradation of the extracellular matrix in cancer invasiveness and metastasis. Very similar to podosomes, invadopodia are found in invasive cancer cells and are ...
, which are important for metastasis. Biotin-based proximity labeling studies demonstrate increased protein tagging of intrinsically disordered regions, suggesting that biotin-based proximity labeling can be used to study the roles of IDRs. A photosensitizer nucleus-targeted small molecule has also been developed for photoactivatable proximity labeling.


Photocatalytic-based Proximity Labeling

A new frontier in the field of proximity labeling exploits the utility of photocatalysis to achieve high spatial and temporal resolution of proximal protein microenvironments.Geri, Jacob; et al. 2020. "Microenvironment mapping via Dexter energy transfer on immune cells". Science.
/ref> This photocatalytic technology leverages the photonic energy of iridium-based photocatalysts to activate diazirine probes that can tag proximal proteins within a tight radius of about four nanometers. This technology was developed by the Merck Exploratory Science Center in collaboration with researchers at Princeton University.Cross, Ryan. 2020. "Merck and Princeton scientists create method to map cell-surface microenvironments". Chemical And Engineering News.
/ref>


References

{{reflist Protein methods Molecular biology techniques