Protein-protein interactions (PPIs) trigger a wide range of biological signaling pathways that are crucial for biomedical research and drug discovery. Various techniques have been used to study specific proteins, including affinity chromatography, activity-based probes, affinity-based probes and photo-affinity labeling (PAL). PAL has become one of the most powerful strategies to study PPIs. Traditional photocrosslinkers are used in PAL, including benzophenone, aryl azide, and diazirine. Upon photoirradiation, these photocrosslinkers (Pls) generate highly reactive species that react with adjacent molecules, resulting in a direct covalent modification. This review introduces recent examples of chemical proteomics study using PAL for PPIs.

Accumulation of abnormal tau aggregates in neuron is an important pathological signature in multiple neurodegenerative disorders including Alzheimer's disease. Tau is a neuron specific microtubule-associated protein that regulates microtubule stability, which is critical for axonal outgrowth and synaptic plasticity. In a pathological condition, tau dissociates from microtubules and forms insoluble aggregates called neurofibrillary tangles (NFTs). The accumulation of NFTs in neuron directly correlates with microtubule dysfunction and neuronal degeneration. Due to the pathophysiological importance of tau, great efforts have been made to understand tau aggregation processes and find therapeutics to halt or reverse the processes. However, progress has been slow due to the lack of a suitable method for monitoring tau aggregation. In this mini-review, we will review the conventional methods for studying tau aggregation, and introduce recent cell-based sensor approaches that allow monitoring tau aggregation in living cells.

Tauopathy is a collective term for neurodegenerative diseases associated with pathological modifications of tau protein. Tau modifications are mediated by many factors. Recently, reactive oxygen species (ROS) have attracted attention due to their upstream and downstream effects on tauopathy. In physiological conditions, healthy cells generate a moderate level of ROS for self-defense against foreign invaders. Imbalances between ROS and the anti-oxidation pathway cause an accumulation of excessive ROS. There is clear evidence that ROS directly promotes tau modifications in tauopathy. ROS is also highly upregulated in the patients’ brain of tauopathies, and anti-oxidants are currently prescribed as potential therapeutic agents for tauopathy. Thus, there is a clear connection between oxidative stress (OS) and tauopathies that needs to be studied in more detail. In this review, we will describe the chemical nature of ROS and their roles in tauopathy.

Abnormal tau aggregation is a pathological hallmark of many neurodegenerative disorders and it is becoming apparent that soluble tau aggregates play a key role in neurodegeneration and memory impairment. Despite this pathological importance, there is currently no single method that allows monitoring soluble tau species in living cells. In this regard, we developed a cell-based sensor that visualizes tau self-assembly. By introducing bimolecular fluorescence complementation (BiFC) technique to tau, we were able to achieve spatial and temporal resolution of tau-tau interactions in a range of states, from soluble dimers to large aggregates. Under basal conditions, tau-BiFC cells exhibited little fluorescence intensity, implying that the majority of tau molecules exist as monomers. Upon chemically induced tau hyperphosphorylation, BiFC fluorescence greatly increased, indicating an increased level of tau-tau interactions. As an indicator of tau assembly, our BiFC sensor would be a useful tool for investigating tau pathology.

Recent evidence suggests that tau aggregates are not only neurotoxic, but also propagate in neurons acting as a seed for native tau aggregation. Prion-like tau transmission is now considered as an important pathogenic mechanism driving the progression of tau pathology in the brain. However, prion-like tau species have not been clearly characterized. To identify infectious tau conformers, here we prepared diverse tau aggregates and evaluated the effect on inducing intracellular tau-aggregation. Among tested, tau dimer containing P301L-mutation is identified as the most infectious form to induce tau pathology. Biochemical analysis reveals that P301L-tau dimer is covalently cross-linked with a disulfide bond. The relatively small and covalently cross-linked tau dimer induced tau pathology efficiently in primary neurons and also in tau-transgenic mice. So far, the importance of tau disulfide cross-linking has been overlooked in the study of tau pathology. Here our results suggested that tau disulfide cross-linking might play critical role in tau propagation by producing structurally stable and small tau conformers.