For scientific researchers studying molecular biology, when they obtain an interesting gene/protein and want to conduct in - depth functional verification, the study of protein - protein interactions is an essential part. Protein - protein and protein - nucleic acid interactions are widespread in various life processes and play important regulatory roles. Developing methods for large - scale analysis of interactions between biomolecules is of great significance for exploring biological functions and disease intervention. Although traditional biochemical research methods such as pulldown, Co - IP, and yeast two - hybrid are widely used to uncover potential protein - protein interactions, they all have their own drawbacks and limitations.

If you encounter the following problems in your study of protein - protein interactions:

· What if there is no good specific antibody for the target protein or there is no corresponding antibody at all?

· What if the interacting proteins obtained by Co-IP do not include the protein you are interested in?

· If you want to escape the cumbersome steps of preparing GST Pull-down samples and avoid false positive results, what should you do?

· If you are concerned about the real protein - protein interactions within cells and don't want to miss any direct or indirect interactions, what should you do?

Then let's take a look at our Proximity-based Labeling (PL for short) technology!

It is an emerging technology for screening protein - protein interactions (PPI), providing an alternative complementary to traditional methods (such as Co - IP, yeast two - hybrid, etc.) for studying protein interactions. Its principle is to fuse an enzyme with proximity - labeling function (biotin ligase) to the bait protein. Through enzyme - catalyzed covalent modification, proteins adjacent to the bait protein are labeled with biotin. Finally, streptavidin magnetic beads are used to enrich the biotin - labeled proteins for mass spectrometry identification. Biotin is a natural coenzyme that can bind tightly to glycoproteins, avidin or similar proteins, and streptavidin. This strong interaction can easily purify and identify the biotin - labeled proteins .

Put simply, in the bustling city of the cell, proteins are like the city's residents. Scientists want to know how these residents (proteins) communicate with each other. But to find clues to this communication, they need a way to mark the residents that are close to each other. Proximity labeling enzymes are like special "detectives" that can identify and mark the protein residents that are close to each other in the cell city. This enzyme has a special ability: it can "sniff out" proteins that are very close and leave a "tag" on them. This tag is like a special piece of clothing for these proteins, allowing scientists to identify and track them using special instruments such as mass spectrometers.

There are many commonly used enzymes with proximity marker functions, such as BioID, APEX, TurboID, etc., which can work in the environment in which proteins live naturally (that is, in cells and organisms), helping us discover those previously unknown protein friendships, so as to better understand how cells work and what changes occur in disease states, and help us understand the interactions between proteins more realistically. Here is also a brief introduction to several very common proximity marker enzymes.

1、BioID & BioID2

BioID is the earliest detective in the cell town and is a mutant (R118G) of biotin ligase (BirA) from Escherichia coli. The ligase catalyzes the formation of biotin - 5'-AMP anhydride, which diffuses out of the active site to biotinylate proximal endogenous proteins on nucleophilic residues such as lysine. BioID is a promiscuous biotin ligase that can effectively label adjacent proteins. BioID2 naturally lacks a DNA - binding domain, is approximately one - third smaller than BioID, and can enhance the targeting and localization of fusion proteins. Compared with BioID, BioID2 requires less biotin to accomplish similar biotinylation, and this property may be beneficial in systems where biotin supplementation is difficult.

2、APEX & APEX2

One year after the advent of BioID, the Alice Ting laboratory demonstrated that APEX, used as a genetic tag for electron microscopy, could be applied to effective proximity labeling. APEX is a 27 kDa monomeric ascorbate peroxidase that can catalyze the oxidation of biotin-phenol to the short-lived biotin-phenoxy radical of \((<1 ms)\) in the presence of \(H_{2} O_{2}\). One of the main limitations of APEX is its low sensitivity. Sensitivity can be increased with higher expression levels, but for many fusion proteins, this leads to protein/organelle aggregation. APEX2 is an improved APEX mutant with higher sensitivity and can be used more effectively for proximity labeling.

3、TurboID、miniTurbo & Split-TurboID

TurboID is a genetically engineered biotin ligase that uses ATP to convert biotin into biotin-AMP, an active intermediate that can covalently label proximal proteins. BioID and BioID2 may take hours to label proteins, while APEX2 can label proteins rapidly but requires the addition of \(H_{2} O_{2}\), which is toxic to living cells. To circumvent these issues, the Ting Lab used yeast-display-based directed evolution to generate two mutants: 35 kD TurboID (with 15 mutations compared to BirA) and 28 kD miniTurbo (with an N-terminal domain deletion and 13 mutations compared to wild-type BirA). Both TurboID and miniTurbo can complete biotin labeling within 10 minutes without any toxicity issues. The signal of TurboID is significantly greater than that of miniTurbo at early time points. miniTurbo is like a mini version of TurboID, it is more compact and can label and detect proteins more precisely.

Split-TurboID is a more advanced detective technique. It divides the labeling ability of TurboID into two parts, similar to a communicator split in half. These two halves are not active when separate, but when they come close to each other and interact within the cell, they can recombine into a complete TurboID, thus activating the labeling function. This method can more precisely control the labeling process, ensuring that labeling occurs only when two proteins are truly close and interacting.

At present, the commonly used proximity labeling enzyme in GenePionner is TurboID. The molecular weight, working temperature, working time, and working range of other commonly used proximity labeling enzymes are also listed in the table for our audience. You can check it by yourself!

 

 

Compared with traditional protein - protein interaction research methods such as pull - down, Co - IP, BiFC, and yeast two - hybrid, the main advantages of PL are:

1、No antibody required (Co-IP requires an antibody)

Co-IP is like at a party, where we use a specific person (a tagged protein) to attract his friends. If two proteins are good friends, when we pull out this person with the antibody of one protein, his friends will also be pulled out together. This method can tell us whether two proteins are good friends, but sometimes it may accidentally pull out some people who are not real friends. When there is no good tag antibody or specific antibody for our target protein, it is very likely to accidentally pull out some "fake friends", while PL usually has a high specificity, reducing the possibility of misjudgment.

2、There is no need to construct a cDNA library, which saves time (required for yeast two-hybrid library construction)

Yeast two-hybrid library construction can be envisioned as a kind of "dating show", specifically designed to explore whether proteins in cells can "successfully pair up". The cDNA library is the "pool of dating guests". Constructing this "guest pool" is a complex and time-consuming process, and it is necessary to ensure its diversity and representativeness to cover all possible protein-protein interactions. If the construction is inadequate, some important interacting proteins may be missed, while proximity labeling does not need to consider so many factors.

3、Weakly and transiently interacting proteins can be identified. (This cannot be achieved by pull - down, Co - IP, BiFC, etc.)

When we obtain interacting proteins through the Co-IP method, but fail to find the proteins we are interested in and expect after rummaging around, where does the problem lie? The problem lies in the fact that the Co-IP method retrieves stable, long-lasting, and firmly interacting proteins. However, those proteins that interact transiently and have weak binding affinity may be overlooked. But you can't say that these transiently interacting proteins with weak binding affinity are insignificant. Just like a mosquito landing on your skin, giving you a gentle bite, and then flying away after you get infected with dengue fever. Can you say that this mosquito is not the main cause of your dengue fever infection? On the contrary, PL can identify such unremarkable transiently acting factors and explain why you got infected with dengue fever.

4、Reflect the real interactions within living cells (Co-IP, pull-down, etc. are in vitro experiments)

Even in complex living cells and animals, PL is like a top detective wearing a "miniature camera" (biotin) sneaking into a crowded party. This "miniature camera" can record the interactions of proteins in their natural communication state, rather than pulling the proteins out of the party scene and observing them artificially in the laboratory. This is something that other methods cannot achieve.

By this point, some of you may be wondering: with this technology being so impressive, for which protein - protein interaction studies can it actually be used? Now, let me summarize the current application scope of PL for you. It can be applied to:

· Study on the interacting proteins of the target protein

· Study on the interacting proteins of disease - critical proteins or drug - target proteins

· Discovery of new markers or target proteins

· Study on Tissue-specific Secretory Proteins

At present, this technology mainly provides convenience for teachers who study mammalian cells, such as teachers from the School of Pharmacy, the School of Traditional Chinese Medicine, the College of Animal Science, and related research institutes. After conducting omics studies such as transcriptomics and proteomics in the early stage, if they want to conduct some follow-up mechanism research, they will choose to use the proximity labeling method. If you, who are reading this article, have a target protein of interest (such as biomarker proteins or drug target proteins obtained in previous studies), you may as well contact us for in - depth cooperation!

The basic experimental procedures of PL include:

1、 Vector construction

Construct the fusion expression plasmid of the target protein - detection tag - turboID. The detection tag is used to verify whether the fusion protein is successfully expressed in cells.

2、Construction of expression cell line

Transfer the vector into the cell line to stably express the fusion protein in the cell line. The cells need to be a cell line model specific to the research content and be capable of conducting transformation experiments. In this experiment, a treatment group and a control group are set up. The treatment group involves the normal expression of the fusion protein, while the control group involves the expression of a control plasmid without the target gene. The purpose is to eliminate the proteins that bind to turboID itself.

3、 Biotinylation treatment

This step is to provide appropriate catalytic conditions to label the proteins adjacent to the target protein with biotin, and then perform affinity purification through streptavidin magnetic beads to enrich the proteins.

4、Protein Mass Spectrometry Detection

Each of the above two groups was set up with 3 biological replicates, and a total of 6 samples were subjected to proteomics detection to analyze and identify the interacting proteins of the target protein.

The above are the general experimental steps for some of the advantages of the proximity labeling technique. The editor has also selected several high-scoring masterpieces from a vast number of papers. Let's take a look at how previous researchers have flexibly applied this technique throughout the research process!

1、Proximity proteomics identifies septins and PAK2 as decisive regulators of actomyosin-mediated expulsion of von Willebrand factor. 2023, Blood, IF=20.3

This article mainly investigated the proximity labeling technique and von Willebrand factor (VWF). The researchers combined the APEX2 proximity labeling technique with an innovative double loss-of-function screen to identify proteins associated with the function of the actomyosin ring. Additionally, methods such as siRNA screening, immunofluorescence, and live-cell imaging were jointly utilized. This is of great significance for understanding how endothelial cells control the hemostasis process and how to treat related diseases by intervening in specific molecular targets.

 

 

After tissue injury, von Willebrand factor (VWF) is rapidly released from Weibel-Palade bodies (WPBs, unique organelles of endothelial cells) into the vascular lumen to recruit platelets. The size of VWF multimers places a huge burden on the secretory mechanism of endothelial cells, and endothelial cells overcome this problem by utilizing myosin rings. The formation or function of myosin rings is a new therapeutic target for thrombosis, so it is necessary to identify the proteins associated with this dynamic process. The specific experimental design includes: (1) Fusing APEX2 with Rab27a protein to selectively target WPBs and biotinylate neighboring proteins; (2) To monitor the changes in neighboring proteins during the fusion of HUVECs with the plasma membrane, unstimulated human umbilical vein endothelial cells (HUVECs) and HUVECs with different secretagogue stimuli (PMA, HAI) were designed. Research shows that 44 proteins common to the two stimuli may be the core proteins of the secretory mechanism. Among them, p21-activated kinase 2 (PAK2) recruits septin hetero-oligomers to control the function of myosin rings, which facilitates extracellular release. Inhibiting PAK2 or septin reduces the efficiency of VWF release, leading to the inability to form platelet-capturing strands. This new molecular mechanism provides a new therapeutic target for controlling thrombotic diseases, and has fundamental and clinical significance for understanding how endothelial cells control hemostasis and, more generally, how myosin controls regulated secretion .

2、Allosteric Activation of Transglutaminase 2 via Inducing an "Open" Conformation for Osteoblast Differentiation. 2023, Advanced Science, IF=15.1

This study was completed by the research team led by Prof. Zeng Kewu/Tu Pengfei from the State Key Laboratory of Natural and Biomimetic Drugs, School of Pharmaceutical Sciences, Peking University. The team discussed the allosteric activation of transglutaminase 2 (TGM2) and how this process promotes osteoblast differentiation. Using the proximity labeling technique, they determined that TGM2 promotes the activation of the downstream mitochondrial energy synthesis pathway.

 

 

Osteoblast-mediated bone formation is an important part of bone metabolism. Abnormal bone metabolism can lead to the occurrence of bone diseases such as osteoporosis. Forskolin (FSK) is a representative active ingredient of the traditional medicinal plant Coleus forskohlii, and it has a certain effect of promoting osteogenic differentiation, indicating its potential therapeutic effect on bone diseases such as osteoporosis. Transglutaminase 2 (TGM2) is a multifunctional enzyme. Some studies have shown that TGM2 can induce osteoblast differentiation, improve bone loss, and inhibit the increase in the number of osteoclasts. However, there is no research report on candidate drugs targeting TGM2 to promote osteoblast differentiation and bone remodeling. In this study, the authors constructed a small molecule activity probe and determined that TGM2 is the direct target of forskolin in inducing osteoblast differentiation. Mechanistic studies have found that forskolin can directly act on the catalytic core domain of TGM2, induce allosteric changes in the target protein, and increase its activity. At the same time, the proximity labeling technique was used to determine that TGM2 promotes the activation of the downstream mitochondrial energy synthesis pathway, promotes osteoblast differentiation, and exerts the anti-osteoporosis effect of forskolin. This study reveals the pharmacological mechanism of the natural active small molecule compound forskolin in regulating bone metabolism, and also provides new ideas for the research and development of new drugs for the treatment of osteoporosis .

3、Snx14 proximity labeling reveals a role in saturated fatty acid metabolism and ER homeostasis defective in SCAR20 disease. 2020, Proc Natl Acad Sci U S A, IF=9.412

This article mainly investigated the role of Snx14 protein in saturated fatty acid metabolism and endoplasmic reticulum (ER) homeostasis, as well as the impact of these functional defects in autosomal recessive spastic ataxia of Charlevoix-Saguenay 20 (SCAR20) disease. The author's team used APEX2 proximity labeling, and the research revealed that Snx14 is associated with the endoplasmic reticulum-lipid droplet.

The protein composition of the (ER-LD) contact site was investigated, and the functional interaction between Snx14 and the Δ-9 fatty acid desaturase SCD1 was defined. Further research was conducted in combination with lipidomics, providing important insights into the molecular mechanism of SCAR20 disease.

 

 

Fatty acids (FAs) are central cellular metabolites that promote lipid synthesis. Dysregulation of FA processing and storage can lead to metabolic and neurological disorders. Saturated lipids (SFA) are particularly harmful to cells, but how the level of lipid saturation is maintained remains poorly understood. This study first discovered that the disease - associated protein Snx14 in spinocerebellar ataxia SCAR20 is an endoplasmic reticulum - lipid droplet (ER - LD) tethering protein and a factor required to maintain the balance of lipid saturation in the cell membrane. SFA can damage the integrity of the endoplasmic reticulum network in SNX14 KO cells. This study constructed a Snx14 proximity - labeling system, revealing the protein composition related to Snx14 at the ER - LD contact sites and identifying the functional interaction between Snx14 and the Δ - 9 FA desaturase SCD1. Proteomics and lipidomics identified the disruption of the ability of FA metabolism and membrane lipid composition in SNX14 - / - cells [6]. Proximity - labeling techniques, as well as proteomics and lipidomics analyses, revealed the role of Snx14 in FA and ER homeostasis, providing insights into the proteins at the ER - LD contact sites and the lipogenic response.

Summary

Together with the editor, we have in - depth studied the Proximity Labeling (PL) technology through literature. Have you found that in the field of protein - protein interaction research, compared with traditional technologies, it indeed brings us convenience:

1. PL can precisely capture the interactions between biomolecules in time and space, especially those weak or transient interactions.

2. PL experiments can be carried out in living cells, allowing researchers to observe the behavior and interactions of proteins under natural physiological conditions. It can also explore the subcellular localization, topological structure, dynamic migration process of membrane proteins, as well as their interacting proteins, which to some extent facilitates the localization and structural analysis of proteins.

3. Compared with traditional protein-protein interaction research methods, PL reduces the complexity of sample preparation and potential sample loss.

4. By revealing the protein - protein interaction network, PL helps to identify new drug targets and provides new strategies for disease treatment.

These advantages make the proximity labeling technique a very powerful tool in the study of protein - protein interactions, especially playing an important role in proteomics and systems biology research. With the development of science and technology, PL itself is also constantly being optimized. For example, by improving the activity of the enzyme, enhancing the labeling specificity, and reducing non - specific binding, etc. Combining it with existing technologies will undoubtedly be able to solve more biological problems.