Science: Using Phosphoproteomics to Elucidate Signaling Pathways for Opioid Activation in the Brain

Opioids are potent painkillers for the brain, but they have a range of harmful 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 in protein phosphorylation--protein molecular switching---patterns in five different regions of the brain and corresponding to the desired and undesirable therapeutic effects of opioids. These results will provide a means to identify new drug targets and design a new class of painkillers with fewer side effects. The related results of this 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 company's chain of command. 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 the 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 cell. In cells, instructions are passed on to other proteins by changing the function of these "cell employees". One way to change function is phosphorylation-attaching a phosphoryl group 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, 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.


Protein activity profiling

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. As a result, they invented a term named phosphoproteomics. In a recent study, they analyzed activation of signaling pathways that respond to opioids in different regions of the brain. To achieve this, they used a recently developed method called EasyPhos.

To understand how opioids work, we must understand their effects on the brain. "With phosphorylation proteomics, we were able to analyze more than 50,000 phosphorylation sites at once and obtained all active signaling pathway maps in brain samples. We found 1000 changes of signaling pathways after exposure to opioids," said Jeffrey Liu, first author of the research 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 phosphoproteomics to measure the level of 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, particularly focused on these cascades of opioids that are 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. This study introduces a new concept for opioids as 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 proteins in the brain are a company, and phosphoproteomics 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 the effects of drugs at a global level 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 phosphoproteomics and predicts that this method can also be used to generate instructions on how cells use their chains to process information and knowledge of the effects of drugs on other organs.

Dr. Lee-Yuan Liu-Chen of the Temples University's Louis Katz School of Medicine and his team used two drugs to conduct behavioral experiments and discovered that they have similar analgesic effects, but their side effects vary widely. These 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.



Liu J J, Sharma K, Zangrandi L, et al. In vivo brain GPCR signaling elucidated by phosphoproteomics. Science, 2018, 360(6395): eaao4927.

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