Sorting out a tangled mess

Research has shown that rogue proteins are responsible for several brain-related illnesses, but how they clump together to cause slow-burn chaos is still not entirely understood. Scientists from the Adolphe Merkle Institute’s BioPhysics group have developed a new technique to track some of those changes.   

Tauopathies are a group of neurodegenerative diseases that affect the way our brain cells communicate among themselves. These disorders, which include Alzheimer's disease, progressive supranuclear palsy (PSP), Parkinson’s disease, and some forms of dementia, are characterized by the clumping of a protein called tau inside the brain cells.

Neurons that help transmit messages and information have long, thread-like extensions called axons, which act like communication highways, allowing information to travel smoothly in the brain. In tauopathies, the tau protein that usually helps support and stabilize the axons’ structure starts to clump together. These clumps, or tau tangles, disrupt the transportation of vital nutrients and molecules along the axons, hindering the smooth flow of information. This leads to problems in the communication between brain cells and can eventually cause them to become damaged or die.

The effects of tauopathies on a person can vary depending on the specific disorder and which parts of the brain are affected. Common symptoms include memory loss, confusion, difficulties with movement and coordination, and changes in behavior or personality. The exact reasons why tau tangles form have yet to be fully understood. Researchers believe that it may be due to a combination of genetic factors and environmental influences.

One approach to investigating the aggregation of the proteins is to fluorescently label them, allowing scientists to identify potential therapeutic targets. Usually, tau proteins would be labeled with a dye that bonds with residues of cysteine, an amino acid. However, these residues are implicated in the aggregation mechanism. To avoid disturbing this process, the AMI researchers developed a different strategy.

Rather than target the cysteine residues, they chose to attach a fluorophore to one of the two ends of the protein, in this case, the so-called C-terminus.  To achieve this, they used a technique known as site-specific protein labeling via sortase-mediated transpeptidation. Derived from bacteria, a sortase is an enzyme that can modify surface proteins by targeting a specific amino acid sequence of a protein.

After binding the fluorophore, the researchers investigated the effects of the modifications on the protein’s secondary structure and compared the aggregation kinetics with those of native tau protein in vitro. They also used transmission electron and atomic force microscopy to compare the resulting tau fibrils’ morphology.  Their results revealed that the native and C-terminally labeled tau proteins exhibited similar properties concerning the secondary structure, fibril morphology, and speed of aggregation.

“We think that our C-terminal labeling strategy of the tau protein may be useful for studies of tau aggregation using single-molecule fluorescence methods with minimal effects on the structure of the native protein conformation,” adds AMI’s chair of BioPhysics, Prof. Michael Mayer.  Therefore, these molecules may be helpful for identifying therapeutic drug candidates that inhibit the formation of toxic tau aggregates.

Reference: Bryan, L.; Awasthi, S.; Li, Y.; Nirmalraj, P. N.; Balog, S.; Yang, J.; Mayer, M. Site-Specific C-Terminal Fluorescent Labeling of Tau Protein. ACS Omega 2022, 7 (50), 47009–47014.