Plastic nanoparticles cause proteome stress and aggregation by compromising cellular protein homeostasis ex vivo and in vivo.

Decomposition of plastic materials into minuscule particles and their long-term uptake pose increasing concerns on environmental sustainability and biosafety. Besides common cell viability and cytotoxicity evaluations, how plastic nanoparticles interfere with different stress response pathways and affect cellular fitness has been less explored. Here, we provided the first piece of evidence to demonstrate plastic nanoparticles potentially can deteriorate proteome stability, compromise cellular protein homeostasis, and consequently cause global proteome misfolding and aggregation. Polystyrene (PS) nanoparticles of different sizes and surface charges were exploited as model plastic materials. In cell lysate and human blood plasma, naked PS nanoparticles with hydrophobic surface deteriorated proteome thermodynamic stability and exaggerated its aggregation propensity. While no cell viability ablation was observed in cells treated with PS nanoparticles up to 200 µg·mL-1, global proteome aggregation and stress was detected by a selective proteome aggregation sensor. Further proteomics analysis revealed how protein homeostasis network was remodeled by positively charged PS nanoparticles via differential expression of key proteins to counteract proteome stress. In mice model, size-dependent liver accumulation of positively charged PS nanoparticles induced hepatocellular proteome aggregation and compromised protein homeostasis network capacity that were invisible to standard alanine transaminase and aspartate transaminase (ALT/AST) liver function as-say and histology. Meanwhile, long-term liver accumulation of plastic nanoparticles deteriorated liver metabolism and saturated liver detoxification capacity of overdosed acetaminophen. This work highlighted the impact of nanoplastics on cellular proteome integrity and cellular fitness that are invisible to current biochemical assays and clinical tests.

Tailoring the Amphiphilicity of Fluorescent Protein Chromophores to Detect Intracellular Proteome Aggregation in Diverse Biological Samples.

The formation of amorphous misfolded and aggregated proteins is a hallmark of proteome stress in diseased cells. Given its lack of defined targeting sites, the rational design of intracellular proteome aggregation sensors has been challenging. Herein, we modulate the amphiphilicity of fluorescent protein chromophores to enable selective detection of aggregated proteins in different biological samples, including recombinant proteins, stressed live cells, intoxicated mouse liver tissue, and human hepatocellular carcinoma tissue. By tuning the number of hydroxyl groups, we optimize the selectivity of fluorescent protein chromophores toward aggregated proteins in these biological samples. In recombinant protein applications, the most hydrophobic P0 (cLogP = 5.28) offers the highest fold change (FC = 31.6), sensitivity (LLOD = 0.1 µM), and brightness (Φ = 0.20) upon binding to aggregated proteins. In contrast, P4 of balanced amphiphilicity (cLogP = 2.32) is required for selective detection of proteome stresses in live cells. In mouse and human liver histology tissues, hydrophobic P1 exhibits the best performance in staining the aggregated proteome. Overall, the amphiphilicity of fluorescent chromophores governs the sensor's performance by matching the diverse nature of different biological samples. Together with common extracellular amyloid sensors (e.g., Thioflavin T), these sensors developed herein for intracellular amorphous aggregation complement the toolbox to study protein aggregation.

Integrated Imaging and Proteomic Sensors Resolve Proteome Aggregation in Liver Caused by Non-steroidal Anti-inflammatory Drug Overdose.

Given the extreme heterogeneity and the loss of defined protein structures, misfolded and aggregated proteins are technically challenging to visualize and analyze. Herein, we assembled an integrated sensor system to resolve aggregated proteome in live cells and animal liver tissues that are overdosed by non-steroidal anti-inflammatory drugs (NSAIDs). A fluorogenic protein aggregation sensor (AggStain) first discovered the presence of aggregated proteome upon overdosing liver cells with NSAIDs. A solvatochromic protein aggregation sensor (AggRetina) further quantified the compactness (polarity) inside these cellular aggregates. Importantly, we exploited a proteomic sensor (AggLink) to selectively capture aggregated proteins upon NSAID overdose and profile their composition, revealing global collapse of cellular protein homeostasis. Finally, we detected subtle proteome aggregation in mouse liver tissue without obvious acute injury at a low NSAID dosage. Overall, we demonstrated an integrated sensor toolset for proteome aggregation studies and unveiled for the first time that NSAID overdose can cause proteome aggregation in liver cells and tissues.

Developing an Affinity-Based Chemical Proteomics Method to In Situ Capture Amorphous Aggregated Proteome and Profile Its Heterogeneity in Stressed Cells.

Stress induced amorphous proteome aggregation is a hallmark for diseased cells, with the proteomic composition intimately associated with disease pathogenicity. Due to its particularly dynamic, reversible, and dissociable nature, as well as lack of specific recognition anchor, it is difficult to capture aggregated proteins in situ. In this work, we develop a chemical proteomics method (AggLink) to capture amorphous aggregated proteins in live stressed cells and identify the proteomic contents using LC-MS/MS. Our method relies on an affinity-based chemical probe (AggLink 1.0) that is optimized to selectively bind to and covalently label amorphous aggregated proteins in live stressed cells. Especially, chaotrope-compatible ligation enables effective enrichment of labeled aggregated proteins under urea denaturation and dissociation conditions. Compared to conventional fractionation-based method to profile aggregated proteome, our method showed improved enrichment selectivity, detection sensitivity, and identification accuracy. In HeLa cells, the AggLink method reveals the constituent heterogeneity of aggregated proteome induced by inhibition of pro-folding (HSP90) or pro-degradation (proteasome) pathway, which uncovers a synergistic strategy to reduce cancer cell viability. In addition, the unique fluorogenicity of our probe upon labeling aggregated proteome detects its cellular location and morphology. Together, the AggLink method may help to expand our knowledge of the previously nontargetable amorphous aggregated proteome.

Covalent Solvatochromic Proteome Stress Sensor Based on the Schiff Base Reaction.

Covalent-type probes or sensors have been seldom reported for aggregated proteins. Herein, we reported a series of covalent solvatochromic probes to selectively modify and detect aggregated proteomes through the Schiff base reaction. Such covalent modification was discovered by serendipity using the P1 probe with an aldehyde functional group, exhibiting enhanced fluorescence intensity and unusually large blue shift upon protein aggregation. Supported by the biochemical and mass spectrometry results, we identified that this probe can modify the lysine residue of aggregated proteins selectively over folded ones via the Schiff base reaction. The generality of designing such a covalent-type probe was demonstrated in multiple probe scaffolds using different model proteins. Finally, we exploited the distinct solvatochromism of P1 after Schiff base linkage with aggregated proteins to visualize the distinct morphology of aggregated proteomes, as well as to quantify the polarity heterogeneity inside it. This work may intrigue the exploration of other chemical reaction types to covalently functionalize aggregated proteins that were difficult to analyze.

Solvatochromic Cellular Stress Sensors Reveal the Compactness Heterogeneity and Dynamics of Aggregated Proteome.

Deterioration of protein homeostasis (proteostasis) often induces aberrant proteome aggregation. Visualization and dissection of the stressed proteome are of particular interest given their association with numerous degenerative diseases. Recent progress in chemical cellular stress sensors allows for direct visualization of aggregated proteome. Beyond its localization and morphology, the physicochemical nature and the dynamics of the aggregated proteome have been challenging to explore. Herein, we developed a series of solvatochromic fluorene-based D-π-A probes that can selectively and noncovalently bind to a misfolded and aggregated proteome and report on their compactness heterogeneity upon cellular stresses. We achieved this goal by variation of the heterocyclic acceptors to modulate their solvatochromism and binding affinity to amorphous aggregated proteins. The optimized sensor P6 was capable of sensing the polarity differences among different aggregated proteins via its fluorescence emission wavelength. In live cells, P6 revealed the cellular compactness heterogeneity in the aggregated proteome upon cellular stresses. Given the combinative solvatochromic and noncovalent properties, our probe can reversibly monitor the dynamic changes in the aggregated proteome compactness upon stress and after stress recovery, suggesting its potential applications in search of therapeutics to counteract disease-causing proteome stresses.

Derivatizing Nile Red fluorophores to quantify the heterogeneous polarity upon protein aggregation in the cell.

Protein aggregation in the cell is often manifested by the formation of subcellular punctate structures. Herein, we modulated the solvatochromism and solubility of Nile Red fluorophore derivatives to quantitatively study the polarity inside pathogenic protein aggregates, revealing structure- and protein-dependent polarity heterogeneity.

Regulation of Fluorescence Solvatochromism To Resolve Cellular Polarity upon Protein Aggregation.

Common solvatochromic fluorophores exhibit a bathochromic fluorescence emission wavelength shift accompanied by intensity attenuation due to the presence of nonradiative decay pathways at the excited state. Such intrinsic but inevitable fluorescence quenching of solvatochromism impedes its applications to faithfully quantify local polarity, especially in a polar environment. Herein, we report a new donor-π-acceptor (D-π-A) type solvatochromic fluorophore scaffold containing a perfluorophenyl group that exhibits both a solvatochromic emission wavelength shift and a controllable emission intensity upon polarity fluctuation. The regulation of fluorescence solvatochromism and colors was achieved by tuning the aryl donors. We exploited such desired solvatochromism of these probes to monitor protein misfolding and aggregation via wavelength shift. Finally, the polarity of pathogenic aggregated proteins was quantified by HaloTag bioorthogonal labeling technology in live cells. While much effort has been devoted to resolving the morphology of pathogenic aggregated proteins, this work provides quantitative hints regarding the chemical information at this disease-related protein interphase.

Derivatizing merocyanine dyes to balance their polarity and viscosity sensitivities for protein aggregation detection.

Protein misfolding and aggregation processes involve local polarity and viscosity fluctuation. Herein we modulated the polarity and viscosity sensitivities of merocyanine dyes to detect protein aggregation. We demonstrated how structural modulation balanced these two fluorescence sensitivities and affected the detection of misfolded and aggregated proteins.

A Solvatochromic Fluorescent Probe Reveals Polarity Heterogeneity upon Protein Aggregation in Cells.

We report a crystallization-induced emission fluorophore to quantitatively interrogate the polarity of aggregated proteins. This solvatochromic probe, namely "AggRetina" probe, inherently binds to aggregated proteins and exhibits both a polarity-dependent fluorescence emission wavelength shift and a viscosity-dependent fluorescence intensity increase. Regulation of its polarity sensitivity was achieved by extending the conjugation length. Different proteins bear diverse polarity upon aggregation, leading to different resistance to proteolysis. Polarity primarily decreases during protein misfolding but viscosity mainly increases upon the formation of insoluble aggregates. We quantified the polarity of aggregated protein-of-interest in live cells via HaloTag bioorthogonal labeling, revealing polarity heterogeneity within cellular aggregates. The enriched micro-environment details inside misfolded and aggregated proteins may correlate to their bio-chemical properties and pathogenicity.

Common Pitfalls and Recommendations for Using a Turbidity Assay to Study Protein Phase Separation.

The turbidity assay is commonly exploited to study protein liquid-to-liquid phase separation (LLPS) or liquid-to-solid phase separation (LSPS) processes in biochemical analyses. Herein, we present common pitfalls of this assay caused by exceeding the detection linear range. We showed that aggregated proteins of high concentration and large particle size can lead to inaccurate quantification in multiple applications, including the optical density measurement, the thermal shift assay, and the dynamic light scattering experiment. Finally, we demonstrated that a simple sample dilution of insoluble aggregated protein (LSPS) samples or direct imaging of liquid droplets (LLPS) can address these issues and improve the accuracy of the turbidity assay.

Quantitative interrogation of protein co-aggregation using multi-color fluorogenic protein aggregation sensors.

Co-aggregation of multiple pathogenic proteins is common in neurodegenerative diseases but deconvolution of such biochemical process is challenging. Herein, we developed a dual-color fluorogenic thermal shift assay to simultaneously report on the aggregation of two different proteins and quantitatively study their thermodynamic stability during co-aggregation. Expansion of spectral coverage was first achieved by developing multi-color fluorogenic protein aggregation sensors. Orthogonal detection was enabled by conjugating sensors of minimal fluorescence crosstalk to two different proteins via sortase-tag technology. Using this assay, we quantified shifts in melting temperatures in a heterozygous model protein system, revealing that the thermodynamic stability of wild-type proteins was significantly compromised by the mutant ones but not vice versa. We also examined how small molecule ligands selectively and differentially interfere with such interplay. Finally, we demonstrated these sensors are suited to visualize how different proteins exert influence on each other upon their co-aggregation in live cells.

Rational Design of Crystallization-Induced-Emission Probes To Detect Amorphous Protein Aggregation in Live Cells.

Unlike amyloid aggregates, amorphous protein aggregates with no defined structures have been challenging to target and detect in a complex cellular milieu. In this study, we rationally designed sensors of amorphous protein aggregation from aggregation-induced-emission probes (AIEgens). Utilizing dicyanoisophorone as a model AIEgen scaffold, we first sensitized the fluorescence of AIEgens to a nonpolar and viscous environment mimicking the interior of amorphous aggregated proteins. We identified a generally applicable moiety (dimethylaminophenylene) for selective binding and fluorescence enhancement. Regulation of the electron-withdrawing groups tuned the emission wavelength while retaining selective detection. Finally, we utilized the optimized probe to systematically image aggregated proteome upon proteostasis network regulation. Overall, we present a rational approach to develop amorphous protein aggregation sensors from AIEgens with controllable sensitivity, spectral coverage, and cellular performance.

Covalent Probes for Aggregated Protein Imaging via Michael Addition.

Covalent chemical reactions to modify aggregated proteins are rare. Here, we reported covalent Michael addition can generally occur upon protein aggregation. Such reactivity was initially discovered by a bioinspired fluorescent color-switch probe mimicking the photo-conversion mechanism of Kaede fluorescent protein. This probe was dark with folded proteins but turned on red fluorescence (620 nm) when it non-covalently bound to misfolded proteins. Supported by the biochemical and mass spectrometry results, the probe chemoselectively reacted with the reactive cysteines of aggregated proteins via covalent Michael addition and gradually switched to green fluorescence (515 nm) upon protein aggregation. Exploiting this Michael addition chemistry in the malachite green dye derivatives demonstrated its general applicability and chemical tunability, resulting in different fluorescence color-switch responses. Our work may offer a new avenue to explore other chemical reactions upon protein aggregation and design covalent probes for imaging, chemical proteomics, and therapeutic purposes.

Monitoring the Dynamics of Proteome Aggregation in Live Cells Using a Solubilized and Noncovalent Analogue of Fluorescent Protein Chromophores.

Stress-induced intracellular proteome aggregation is a hallmark and a biomarker of various human diseases. Current sensors requiring either cellular fixation or covalent modification of the entire proteome are not suitable for live-cell applications and dynamics study. Herein, we report a noncovalent, cell-permeable, and fluorogenic sensor that can reversibly bind to proteome amorphous aggregates and monitor their formation, transition, and clearance in live cells. This sensor was structurally optimized from previously reported fluorescent protein chromophores to enable noncovalent and reversible binding to aggregated proteins. Unlike all previous sensors, the noncovalent and reversible nature of this probe allows for dynamic detection of both the formation and clearance of aggregated proteome in one live-cell sample. Under different cellular stresses, this sensor reveals drastic differences in the morphology and location of aggregated proteome. Furthermore, we have shown that this sensor can detect the transition from proteome liquid-to-liquid phase separation to liquid-to-solid phase separation in a two-color imaging experiment. Overall, the sensor reported here can serve as a facile tool to screen therapeutic drugs and identify cellular pathways that ameliorate pathogenic proteome aggregation in live-cell models.