Using Phosphorylation Proteomics to Elucidate Signaling Pathways Activated by Opioid in the Brain

Opioids are potent analgesics for brain, but they have a series of side effects, including addiction. In a new study, researchers from the Max Planck Institute for Biochemistry (MPIB) in Germany, the University of Innsbruck in Austria, the University of Innsbruck, the University of the United States Temple University and the University of Copenhagen in Denmark developed a tool to gain a deeper understanding of the brain's response to opioids. They used mass spectrometry to determine changes on protein phosphorylation patterns in five different regions of the brain, and correspond to the desired and undesirable therapeutic effects of opioids. These results provide a means to identify new drug targets and design a new class of painkillers with fewer side effects. The results of the study were published in the June 22, 2018 issue of Science, titled In vivo brain GPCR signaling elucidated by phosphoproteomics.

The cascade of signals that cells use to respond to external stimuli is similar to the command chain of a company. Activation of a receptor (as compared to the company's leadership) provides instructions to other proteins in the cell (as a group of subordinates). This information is passed through the signal cascade of other interacting proteins to lower levels of tissue structure. Just like employees who perform different tasks to keep the company running, proteins are also molecular machines that perform most of the functions of the cells. In cells, instructions are passed to other proteins by changing the function of these "cell employees." One way to change function is to "phosphorylate", attaching a phosphate molecule to a protein. By simultaneously analyzing all of these molecular switches, it is possible to determine the signaling pathway activity in cells or organs. Compared to the study of DNA, the almost identical genetic "blueprint" in all cells, studying this chain of conduct provides a more accurate picture of what is currently happening within the cell.

Matthias Mann, director of the Max Planck Institute for Biochemistry, and his team used mass spectrometry (a method for determining the identity and quantity of proteins in a sample) to describe the phosphorylation patterns of thousands of proteins in many organ samples. In this way, they invented the term “phosphoproteomics”. In a recent study, they analyzed activation of signaling pathways that respond to opioids in different regions of the brain. To achieve this goal, they used a recently developed method called EasyPhos.

In order to understand how opioids work, one must understand their effects on the brain. "The use of phosphorylated proteomics allows us to analyze more than 50,000 phospho sites at once and to obtain a map of all active signaling pathways in brain samples. We found 1000 after exposure to opioids," said Jeffrey Liu, first author of the paper. Changes in multiple signaling pathways have shown a global impact of these drugs on signaling pathways in the brain. "Previous methods did not capture protein phosphorylation on a large scale and missed many important open or closed signaling pathways.

Phosphorylation Proteomics - A Versatile Tool

Liu said, "In our study, we studied the activation of signaling pathways in the brain responsible for the effects of opioids on pain relief. Conversely, parallel activation of other signaling pathways can lead to adverse side effects."

These researchers used phosphorylated proteomics to measure the activity of these signaling pathways that lead to beneficial effects and side effects. Christoph Schwarzer, who collaborated with Liu and Mann at Innsbruck Medical University to focus on this study, focused on these cascades of opioids activated in the brain. During the development of new drugs, these data can be used to identify potential substances with powerful therapeutic effects and fewer side effects. In addition, this study also shows the prospect of reducing side effects by interfering with cascaded signals. Therefore, this study introduces a new concept for opioids. Current opioids are potent painkillers, but they quickly lead to addiction. Therefore, there is an urgent need for new non-addictive opioids.

Imagine that the protein in the brain is a company, and phosphorylation proteomics allows these researchers to focus on the activities of all employees at once, rather than focusing on a few people. Mass spectrometry can be a powerful tool for studying drug targets in the brain or other organs. Mann said: "In the United States, the current epidemic-related death epidemic is a shocking example of the potential consequences of prescription drugs having strong side effects such as addiction. Through mass spectrometry, we are able to understand at a global level. Drug effects and simplify the development of new drugs with fewer side effects.” Mann explained that the design of new drugs is only one of many potential applications for phosphorus proteomics, and he predicted that this method could also be used to generate commands on how cells used them to process information and knowledge of the effects of drugs on other organs.

Dr. Lee-Yuan and Liu-Chen from the Temples University of Louis Kaz, and his team used two drugs to conduct behavioral experiments and found that they have similar analgesic effects, but their side effects vary widely. The researchers analyzed the phosphorylated proteome differences in the brains of the two drug-treated animals and found that these differences were derived from a small number of signaling pathways. Inhibiting one of these signaling pathways can greatly reduce some side effects.