Potential diagnostic tool finds protein clumps in living cells
Chemical tag distinguishes Parkinson’s cells from healthy ones
Researchers have developed a chemical tag that can detect and measure protein clumping, which is thought to contribute to neurodegenerative diseases like Parkinson’s, in living cells.
The potential diagnostic tool, called TME, readily distinguished living white blood cells derived from Parkinson’s patients over those from healthy individuals, outperforming current methods that rely on killing cells before testing, the researchers reported in a study.
“With our TME chemical tag, researchers can now analyse the proteins’ behaviour directly in living cells — critical information for Parkinson’s disease and other neurodegenerative diseases,” Yuning Hong, PhD, the study’s lead and an associate professor at La Trobe University in Australia, said in a university news story.
The study, “Global analysis of endogenous protein disorder in cells,” was published in Nature Methods.
Proteins are long chains of amino acid building blocks that fold into precise shapes to carry out their function. Cells have a stringent quality-control system to ensure proteins are correctly folded, and those that are misfolded are quickly degraded and cleared. Impairment of this system can result in the accumulation of unfolded proteins that are susceptible to clumping, or aggregation. This can disrupt cell function or cause cell death.
Diagnostic tool looks at unfolded proteins
In Parkinson’s, clumps of alpha-synuclein protein are thought to contribute to the progressive death of brain cells responsible for making the signaling molecule dopamine. Disease symptoms arise from a lack of dopamine signaling.
“In nearly 85 per cent of cases of Parkinson’s disease the causes are unknown, but we do know that abnormal clumps or ‘aggregates’ of these proteins are a marker of the disease in its advanced stages,” Hong said.
Such protein clumping is also associated with various other neurodegenerative disorders, including Alzheimer’s disease, Huntington’s disease, and some non-neurological conditions.
To analyze the behavior of disordered proteins using current methods, scientists must kill the cells, which may change the protein’s natural state. “Not much is known about these proteins as previously they couldn’t be identified in live cells using traditional methods,” Hong said.
“Disordered proteins are highly dynamic and heterogenous — their structure changes shape depending on their environment and killing the cell could alter their behaviour,” said Shouxiang Zhang, PhD, the study’s first author and a researcher at La Trobe.
To address these limitations, the team developed a method based on a chemical probe, TME, to capture the state of unfolded proteins in living cells.
TME is designed to react with amino acids, protein’s building blocks, usually buried within a correctly folded protein, which are exposed when a protein misfolds. Reaction with unfolded proteins, but not folded proteins, triggers a fluorescent signal that can be monitored within living cells.
To enrich and quantify TME-labeled unfolded proteins, the team developed a workflow dubbed Reactive Unfolded protein Based Identification of Cysteine On eNrichment, or RUBICON. This method can also detect some proteins typically found at low cell levels, “which are often overlooked by conventional methods, thereby providing hidden information of … biomarkers and potential drug targets,” the team wrote.
To assess the system’s utility as a potential diagnostic tool, researchers applied TME and RUBICON to immune cells derived from people with Parkinson’s and age- and sex-matched healthy individuals. Tests showed that the TME-based strategy distinguished the two groups more effectively than standard methods that kill cells.
The team applied TME to a Huntington’s disease mouse model to investigate how disease-related misfolded proteins affect cellular protein production. They discovered that affected cells adopted a quality-control mechanism that traps disordered proteins in clusters.
“What leads to the proteins forming aggregates in the first place?” Hong asked. “Is it protective or is it detrimental to the cells? Using TME, we hope that researchers can answer these questions and design new treatments based on what they learn by observing the proteins’ behaviour in live cells over time.”
Hong and colleagues hope to create similar tests to help scientists investigate other neurological and non-neurological diseases.
“More than 50 human diseases have been linked to abnormal protein behaviour and disordered proteins, including Alzheimer’s disease, cystic fibrosis, type 2 diabetes, and certain cancers,” Hong said. “It was clear that there was a need in disease research for the kind of test we have developed and we hope in the future it can help scientists uncover more about the role of disordered proteins in a wide range of diseases.”
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