Development of a new labeling approach for the identification of exosome shipments

Our cells are constantly communicating, and scientists have developed an efficient way to discover the messages they send in biological suitcases filled with proteins called exosomes.

These spherical exosomes, which reside in the inner membrane of a cell but will eventually make their way to another cell, carry large molecules like proteins, a building block of the body and engines of biological activity, and the RNA, which produces proteins.

“It’s an ongoing process,” says Dr. Sang-Ho Kwon, a cell biologist in the Department of Cell Biology and Anatomy at Augusta University’s Medical College of Georgia, and there are growing more evidence that it occurs in both health and disease states.

“We’re trying to figure out this puzzle of what exosomes are doing in different scenarios,” Kwon says. He is the corresponding author of a study in the Extracellular Vesicles Journal detailing a tagging technique he and his research team have developed to analyze the contents of exosomes from any specific cell type to better understand their role in wellness and disease.

“Their contents can help us tell what our cells are telling themselves,” Kwon says, and likely provide early clues that we’re getting sick and help us better understand how we get sick.

Cargo is thought to be loaded early in the formation of exosomes by their precursor endosomes, near the cell membrane, which function much like filling the mail truck at the post office before it sets off. The exosomes will stay there until they are released by the cell to travel to other cells.

Kwon and his team wanted to catch the cargo early in the process.

Currently, the main way to study the contents of exosomes is to first take the exosomes out of their context, to isolate them, a rather laborious process that can lead to inconsistent results. In fact, it can isolate a different type of vesicles, basically from biological compartments in our body of which exosomes are just one type.

The MCG team has developed a more efficient method that allows studying only the contents of exosomes, and studying where they are.

Their tagging system includes a variant of APEX, or ascorbate peroxidase, which is fused to another protein known to search for exosomes. “APEX is a kind of missile that takes me inside,” Kwon says. APEX has a strong affinity for biotin, a B vitamin, which binds to neighboring proteins, such as those carried by the developing exosome, marking them and thus helping to identify them. Biotin can also cross the cell membrane behind which the exosomes are located. Another protein, streptavidin, which naturally binds to biotin, allows them to purify and clearly identify the protein cargo as well as the RNA that will produce the future proteins, using analyzes provided by spectrometry of mass.

Kwon focuses on kidney damage, and they used their system to show that oxidative stress, a byproduct of oxygen utilization, which is excessive and destructive in disease states, alters the cargo content of exosomes made by kidney cells and found in urine. . For example, the expression levels of some proteins changed, and some proteins even disappeared.

Their technique should facilitate the development of databases of the usual contents of a variety of different cell types that will allow comparative studies of what happens to their contents in different disease states such as studies of Kwon’s kidney damage or the cancer.

“It turns out that by looking at exosomes in urine or blood, and looking at what’s inside, we can tell if the cell is injured or healthy,” he says.

Their first use of the labeling system was in live kidney cells in culture. They now want to use it in an animal model of kidney disease.

The science team says the labeling system can further help track changes in exosome content over time and potentially how cells respond to treatment when diseased.

Exosomes are known to play a key role in cell communication, both between cells of the same type and with other types. Again, there is growing evidence of the role exosomes play in disease, including sharing with other cells the news that they are sick and potentially helping to spread disease. “It’s not just about delivering good news. it also conveys bad news,” Kwon says.

He notes that their cargo undoubtedly varies in these various scenarios, an important reason for being able to detect what the exosomes are carrying. The changes may ultimately serve as a good way to monitor response to treatment, another aspect of exosome research that is “exploding,” Kwon says. Scientists are also exploring the possibility of using exosomes to actually deliver treatment, filling these biological packages with drugs that can be delivered directly to the desired location.

In fact, immune cells, which are essential for health and disease, also release exosomes. These biological compartments also seem to play an important role in the elimination of cellular debris and other waste products from the cells.

“It’s an emerging area right now,” Kwon said. Proteins are the main occupant because they can send signals, but they can also bind to other proteins and change their function, he says. RNA can do the same, and tiny microRNAs can alter gene expression and, therefore, cellular function.

Kwon’s interest in exosomes was sealed when, as a postdoc at the University of California, San Francisco, he cultured kidney tubules, which return vital nutrients to the blood and eliminate unwanted ones in the urine, in a dish and found evidence that exosomes played a key role. role in the evolution of gene dynamics there.

He calls the focus on exosomes “reverse science,” with most people looking at how the cell changes while he and a growing number of colleagues look at the packets the cell sends to understand what the cell is doing. While that might not seem like it to most people, he says it’s actually a less complex way to look at cellular activity because you’re looking at a smaller package with a lot less protein.

Reference: Lee BR, Lee TJ, Oh S, et al. Ascorbate peroxidase-mediated in situ labeling of proteins in secreted exosomes. J Extracellular vesicles. 2022;11(6). doi: 10.1002/jev2.12239

This article was republished from the following documents. Note: Material may have been edited for length and content. For more information, please contact the quoted source.

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