Twist Bioscience Oligo Pools Help Understand the Signaling Cascades in T-cells

Inside of any human cell is a chaotic network of chemical and biophysical reactions making up both your metabolism and cellular processes. Much of this activity is performed by proteins. In order for these reaction networks to function effectively, a communication system exists within the cell. To send a cascade of messages to, through, or between metabolic networks, chemical “post-translational” modifications are added to proteins to control their activity or behavior. By far the most common modification is phosphorylation, performed by an enzyme family called “kinases.” Specific kinases control phosphorylation of specific proteins to prevent cross-talk. Here, researchers used Twist Bioscience Oligo Pools to probe the underlying mechanisms of kinase specificity, offering significant insight to our understanding of how our immune systems are controlled.

 

Our cells are remarkable. Billions of these minute units of life come together to form you–a very complex organism. In order to keep you breathing, thinking, laughing, loving and everything else it means to be human; a complex, microscopic chemical world is working tirelessly inside each of your cells.

Peer beneath the fatty outer layer of a human cell and a complex network of membranes, organelles, chemicals, genetic elements and proteins are coordinating with one another to both provide our metabolism, and allow the cell to contribute to its constituent tissue.

And while these parts are able to function in unison, it’s not exactly a well-oiled factory. Instead, the environment inside of a cell is in a state of highly organized chaos, a bit like a major city in rush hour. Take a look at Roche’s comprehensive, interactive “Metabolic pathways” and “Cellular and Molecular Processes” charts to explore just how complex this network of reactions and cellular activity is.

 

Beijing

Beijing, one of the busiest cities in the world is a highly organized, but chaotic metropolis. The processes that make the city work are not dissimilar to the way our cells organize their complex metabolisms and processes. 

The majority of these processes are controlled by the action of proteins. Enzymes are catalysts - the drivers behind nearly all of the chemical reactions in the cell. However, our metabolisms are also dynamic, able to respond accordingly to shifts in the environment or even signals from other cells. When a cell senses change, there are other proteins that exist whose role is to simply to pass on the message. Cascades of communication ripple between these proteins, eventually modifying cellular processes or gene expression, allowing the cell to respond effectively to its situation.

One way these messages are passed involves post-translational protein modification. Such modifications are usually small chemical add-ons that are attached to proteins by the action of other proteins. Of all the modifications, the reversible phosphorylation of tyrosine, serine or threonine amino acids is by far the most common. It’s estimated that approximately 230,000 phosphorylation sites exist in each of our cells, providing a finely detailed network of control. Typically, the phosphorylation of proteins will act as an “on” or “off” switch, either stopping or kick-starting its activity.

Protein phosphorylation is performed by a family of proteins called “protein kinases,” which take a phosphate molecule off of ATP (the energy storage of the cell) and add to it a target amino acid in a specific protein. For these proteins, the avoidance of cross-talk is essential to ensure that only the correct response occurs following signal transduction. Often, a single kinase has a very specific target protein, and will have a single specialized role in a single cascade.

 

Phosphorylation

 

Phosphorylation is the addition of a phosphate molecule to an organic compound. Public domain image source: Wikicommons.

It is not fully understood how the majority of kinases control their specificity on the molecular level. In a recent paper published in eLife, Neel Shah, PhD, and co-workers from the University of California, Berkeley, aimed to use the latest in high quality synthetic DNA technologies to gain a deeper understanding into how protein kinase specificity works.

Their focus was the tyrosine kinases, specifically two kinases: Zap70 and Lck. These kinases play an essential role in Helper T-cell activation. When a naïve membrane-bound T-cell receptor protein is presented with an antigen, huge morphological and chemical responses take place in the cell that differ depending on the type of antigen encountered. Ultimately this cascade defines how the Helper T-cell will mature to contribute to our immune system’s arsenal (see below). A detailed breakdown of the activation cascade can also be explored interactively on the Cell Signaling Technology website.

Helper

 

A basic overview of T-cell activation. When a naïve T-cell is shown an antigen by an Antigen Presenting Cell (APC), a cascade of protein activation brings about huge changes to both T-cell morphology and gene expression. Ultimately these changes mature T-cells into integral parts of our immune system. 

Switching On & Off

Both Zap70 and Lck play a part in the very beginning of this signalling cascade. The authors of the eLife article explain that when the T-cell receptor binds an antigen, Lck is able to then phosphorylate the part of the membrane-bound receptor that reaches inside of the cell. Zap70 is then able to bind to the phosphorylated T-cell receptor. However, at this point its own kinase activity is switched off. Lck then phosphorylates Zap70, switching it on. Once activated, Zap70 phosphorylates other parts of the response cascade, including other kinases, which in turn activate further downstream parts of the cascade, and so on. A wave of protein modification sparks through the cell, ultimately bringing about the required morphological and metabolic changes in response to a given antigen.

Importantly, Zap70 is tightly controlled in this cascade. It is a kinase, however it can neither activate a T-cell receptor nor itself. If it did, the cascade would always be switched on, and an uncontrolled immune response would ensue. Despite having the same core enzyme activity–adding a phosphate to a tyrosine residue of a protein–Lck and Zap70 have to be highly specific for their targets to ensure the correct response.

The authors of the eLife paper continue to explain that contemporary schools of thought hypothesize that kinases have two distinct domains. One part of the protein is solely responsible for kinase activity, and another part is solely responsible for specific substrate binding. As the kinase domains have very weak activity, it is thought that the substrate binding domain must first recognize and bind to its specific target to provide the kinase domain its highly targeted phosphorylation activity. Using a high throughput assay developed by the authors’ research group that tests the phosphorylation activity of specific kinases, the researchers looked deeper into how specificity is controlled.

Negative Charges

Before publishing this paper, the researchers constructed a small library of peptides containing known phosphorylation sites for Lck and Zap70. They found that Lck could phosphorylate peptides that Zap70 couldn’t. Specifically, the tyrosines that were phosphorylated by Zap70 are surrounded by negatively charged amino acids, which make Lck highly inefficient. This was a surprising finding as it suggested that kinase domains themselves are directly involved in mediating specificity.

Crystal structure

 

Zap70

 

Crystal structures of the proteins Lck (top) and Zap70 (right). Image sources: Wikicommons (left) and Wikicommons (bottom). Both Images are licensed under CC BY-SA 3.0

To corroborate their previous findings, in their current study, the authors tested whether this same kinase-controlled specificity holds true across large numbers of target peptides, as well as other kinases closely related to Lck and Zap70. By using Twist Bioscience’s silicon platform for high throughput, high quality, oligonucleotide synthesis, the researchers synthesised 2,600 peptide sequences. Each peptide has 15 amino acids, and containing a specific tyrosine phosphorylation site known to exist in human proteins. Importantly, there was nothing in the peptides that the substrate-binding portion of each kinase could bind to, so the library only tested the specificity of the kinase domain.

As with their previous findings, they found that across the library Lck and Zap70 have bimodal preferences–some peptides in the library get highly phosphorylated, while some peptides show negligible phosphorylation. Importantly, there was a sharp contrast between the types of peptides phosphorylated by Lck and Zap70, again with Zap70 having a strong preference for negatively charged residues surrounding the tyrosine, and Lck being inhibited by these same residues.

By then undertaking analysis of the protein structure of Lck and by testing other kinases closely related to Lck on the same library of peptides, it was shown that the proteins’ specificity are all controlled to some degree by subtle changes in electrostatic forces around the target phosphorylation site. The authors suggest that these subtle changes help afford the tight specificity seen in the kinases, while allowing for simple adaptation to new substrates during evolution of new function.

The findings from this study are important for many reasons. Thanks to the provision of high quality oligonucleotides, their phosphorylation assay platform could be applied in high throughput to understand the activity of protein kinases on vast numbers of potential targets. Therefore the same assay could be used for future studies to understand lesser known cell components. It also shows that kinases are more complex than previously thought. Instead of just the activity of a target domain, both domains likely work together to provide a double-check system affording the exquisite specificity seen in many protein kinases.

Finally, looking at the bigger picture, such high throughput assays, that utilizing the benefits of high-volume and high quality synthetic DNA to probe the complex world of cell signalling, have important implications for human health. For example, fully understanding how T-cells respond when presented with an antigen is of great immunological interest as it can provide new insights into how our bodies respond to disease or allergens, allowing for better treatment. It will also offer greater insights into how our bodies respond to vaccines, allowing for better immunization against potentially deadly diseases in the future. Finally, as mentioned at the start, inside our cells is organized chaos. However, being able to understand that chaos could better equip us with the tools and knowledge to fix things when that chaos becomes disorganized, causing life-threatening diseases like cancer.

 

Featured image: immune system cells attacking an HIV virus. Adobe stock