Visualizing the Multistep Process of Protein Aggregation in Live Cells

Abstract

Conspectus Protein aggregation is a biological phenomenon in which aberrantly processed or mutant proteins misfold and assemble into a variety of insoluble aggregates. Decades of studies have delineated the structure, interaction, and activity of proteins in either their natively folded structures or insoluble aggregates such as amyloid fibrils. However, a variety of intermediate species exist between these two extreme states in the protein folding landscape. Herein, we collectively term these intermediate species as misfolded protein oligomers, including soluble oligomers and preamyloid oligomers that are formed by unfolded or misfolded proteins. While extensive tools have been developed to study folded proteins or amyloid fibrils, research to understand the properties and activities of misfolded protein oligomers has been limited by the lack of methods to detect and interrogate these species in live cells. In this Account, we describe our efforts in the development of chemical methods that allow for the characterization of the multistep protein aggregation process, in particular the misfolded protein oligomers, in living cells. As the start of this journey, we attempted to develop a fluorogenic method wherein the misfolded oligomers could turn on the fluorescence of chemical probes that are conjugated to the protein-of-interest (POI). To this end, we produced a series of destabilized HaloTag variants, formulating the primary component of the AgHalo sensor, which misfolds and aggregates when cells are subjected to stress. When AgHalo is covalently conjugated with a solvatochromic fluorophore, misfolding of the AgHalo conjugate would activate fluorescence, resulting in the observation of misfolded oligomers. Following this work, we extended the scope of detection from AgHalo to any protein-of-interest via the AggTag method, wherein the POIs are genetically fused to self-labeling protein tags (HaloTag or SNAP-tag). Focusing on the molecular rotor-based fluorophores, we applied the modulated fluorescent protein (FP) chromophore core as a prototype for the AggTag probes, to enable the fluorogenic detection of misfolded soluble oligomers of multiple proteins in live cells. Next, we further developed the AggTag method to distinguish insoluble aggregates from misfolded oligomers, using two classes of probes that activate different fluorescence emission toward these two conformations. To enable this goal, we applied physical organic chemistry and computational chemistry to discover a new category of triode-like fluorophores, wherein the π orbitals of either an electron density regulator or the donor–acceptor linkages are used to control the rotational barriers of fluorophores in the excited states. This mechanism allows us to rationally design molecular rotor-based fluorophores that have desired responses to viscosity, thus extending the application of the AggTag method. In summary, our work allows the direct monitoring of the misfolded protein oligomers and differentiation of insoluble aggregates from other conformatio...

Protein Fibrillation under Crowded Conditions

“…For more than 40 years, scientists have characterized the effects of the cellular interior on protein function. The advances in technology, such as in-cell NMR [ 9 , 99 ], single-cell mass spectrometry [ 100 ], fluorescence [ 8 , 10 , 101 , 102 ], FRET [ 103 ] and flow cytometry [ 104 ], electron microscopy [ 5 , 6 , 7 ], and cryo-electron tomography (Cryo-ET), allow scientists to characterize the protein and the protein aggregate structure and function in cells. Cryo-ET is particularly effective at structural characterization of the neurotoxic aggregates [ 105 , 106 , 107 ].…”

“…In the cells and extracellular matrices, the proteins fold and misfold in a crowded environment, surrounded by a complex (and nonrandom) mixture of other solutes [ 3 , 4 ]. Ideally, fibrillation would primarily be studied in living organisms [ 5 , 6 , 7 , 8 , 9 , 10 ], however, measuring the kinetics of fibrillation in the cells poses obvious technical challenges. Instead, researchers have attempted to mimic the crowded environment of cells in vitro, via crowding agents [ 11 ].…”

Xanthone-based solvatochromic fluorophores for quantifying micropolarity of protein aggregates

“…10,11 To explore mechanistic details of protein misfolding and aggregation, Liu [12][13][14] and our group [15][16][17] developed a series of fluorogenic tools based on fluorescence activation. These probes respond to changes in both polarity and viscosity during protein aggregation (Figure 1A, left panel) [18][19][20] , which mostly featured properties based on viscosity sensitivity. Increased viscosity in the local microenvironment of the protein aggregate restricts the internal rotation of fluorophores, resulting in fluorescence enhancement (Figure 1A, left panel).…”

“…These probes respond to changes in both polarity and viscosity during protein aggregation (Fig. 1A, left panel), 18–20 which mostly featured properties based on viscosity sensitivity. Increased viscosity in the local microenvironment of the protein aggregate restricts the internal rotation of fluorophores, resulting in fluorescence enhancement (Fig.…”

Covalent Solvatochromic Proteome Stress Sensor Based on the Schiff Base Reaction

“…Panels of different non-covalent probes and sensors have been designed to illuminate the complex misfolded and aggregated conformations, − including unfolded proteins, misfolded oligomers, − amyloid aggregates, − and amorphous aggregates. − Compared with the non-covalent probes targeting aggregated proteins, covalent probes were still underdeveloped due to the lack of chemical strategies to explore the reactivity inside the misfolded and aggregated proteins. Recently, the Hong and Hatters group first demonstrated that unfolded proteomes can be targeted by globally labeling the exposed cysteine residues via maleimide chemistry and AIEgens upon stress-induced proteome unfolding in live cells. , Echoing Hong’s discovery, our group revealed that cysteine thiols were generally activated upon protein aggregation and can be covalently labeled by 1,4-Michael addition using a color-switch probe .…”