Unraveling Intracellular Protein Corona Components of Nanoplastics via Photocatalytic Protein Proximity Labeling.

Bioaccumulation of nanoplastic particles has drawn increasing attention regarding environmental sustainability and biosafety. How nanoplastic particles interact with the cellular milieu still remains elusive. Herein, we exemplify a general approach to profile the composition of a "protein corona" interacting with nanoparticles via the photocatalytic protein proximity labeling method. To enable photocatalytic proximity labeling of the proteome interacting with particles, iodine-substituted BODIPY (I-BODIPY) is selected as the photosensitizer and covalently conjugated onto amino-polystyrene nanoparticles as a model system. Next, selective proximity labeling of interacting proteins is demonstrated using I-BODIPY-labeled nanoplastic particles in both Escherichia coli lysate and live alpha mouse liver 12 cells. Mechanistic studies reveal that the covalent modifications of proteins by an aminoalkyne substrate are conducted via a reactive oxygen species photosensitization pathway. Further proteomic analysis uncovers that mitochondria-related proteins are intensively involved in the protein corona, indicating substantial interactions between nanoplastic particles and mitochondria. In addition, proteostasis network components are also identified, accompanied by consequent cellular proteome aggregation confirmed by fluorescence imaging. Together, this work exemplifies a general strategy to interrogate the composition of the protein corona of nanomaterials by endowing them with photooxidation properties to enable photocatalytic protein proximity labeling function.

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.

Solvatochromic sensors detect proteome aggregation in stressed liver tissues with hepatic cancer and cirrhosis.

Protein misfolding and aggregation involve complex cellular processes with clinical implications in various diseases. However, the detection of aggregated proteomes without defined 3-D structures in a complex biological milieu is challenging. This study utilizes chromone scaffold-based environment-sensitive fluorophores P1 and P2 to detect misfolded and aggregated proteome in stressed liver cells and the liver tissues diseased patients. The reported crystallization induced emission probes (P1 and P2) exhibit both polarity and viscosity sensitivity, with emission intensity and wavelength linearly correlated to viscosity and polarity. Meanwhile, P1 and P2 selectively and generally fluoresce upon binding to various aggregated proteins. In hepatic cells, P2 outperforms P1 in detecting stress-induced global proteome aggregation. In mouse liver tissue upon drug-induced injury, the fluorescence intensity of P2 correlated with the severity of liver injury, serving as an earlier indicator for liver stress prior to ALT/AST increase. The quantification of emission wavelength reveals lower micro-environmental polarity in liver-injury tissue. In patient-derived tissues with hepatic cancer and cirrhosis, P1 and P2 also report on the presence of aggregated proteome. Together, the reported solvatochromic proteome aggregation sensors can detect hepatic proteome aggregation and analyze its local polarity in cultured cell lines, animal model tissues, and human clinical samples.

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.

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.

A Novel Virus Detection Strategy Enabled by TR512-Peptide-Based Bioorthogonal Capture and Enrichment of Preamplified Nucleic Acid.

High-cost viral nucleic acid detection devices (e.g., qPCR system) are limited resources for developing counties and rural areas, leading to underdiagnosis or even pandemics of viral infectious diseases. Herein, a novel virus detection strategy is reported. Such detection method is enabled by TR512-peptide-based biorthogonal capture and enrichment of commercially available Texas red fluorophore labeled nucleic acid on the functionalized paper. The GST-TR512 fusion protein electrostatically immobilized on the paper is constructed to retain the binding affinity of TR512-peptide toward Texas red fluorophore labeled nucleic acid released in the preamplification process, then the enrichment of analytes enhances fluorescence signal for rapid detection as volume of sample filters through the paper. The method is generally applicable to different nucleic acid preamplification strategies (PCR, RAA, CRISPR) and different virus types (Hepatitis B virus (HBV), African swine fever virus (ASFV), human papillomavirus (HPV), and severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2 or 2019 nCoV)). Finally, a full-set virus detection device is developed in house to detect the presence of Hepatitis B virus (HBV) viral gene in patients' blood samples. Taken together, we first apply TR512-peptide in the signal enrichment and the novel detection strategy may offer an inexpensive, rapid, and portable solution for areas with limited access to a standard diagnosis laboratory.

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.

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.

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.

Wedelolactone alleviates acute pancreatitis and associated lung injury via GPX4 mediated suppression of pyroptosis and ferroptosis.

Acute pancreatitis (AP) is an inflammatory disorder associated with multiple organ failure. Pyroptosis and ferroptosis are two newly recognized cell death, and whether pyroptosis and ferroptosis are involved in AP remain largely elusive. The nature compound Wedelolactone (Wed) exhibits strong anti-inflammatory and antioxidant activities, the present study aims to investigate the effect of Wed on AP and unravel whether Wed could protect against AP and relevant lung injury against pyroptosis and ferroptosis. Our results showed that the pyroptosis inhibitor disulfiram or ferroptosis inhibitor ferrostatin-1 significantly alleviated AP and associated lung injury in the taurocholate or caerulein-induced murine AP model. Administration with Wed ameliorated AP and lung injury as evidenced by improved pathological injuries, reduced serum pancreatic digestive enzymes, and proinflammatory cytokines. The in vivo and in vitro data demonstrated that Wed broadly inhibited caspase1/caspase11 activation, reduced mature interleukin-1ß (IL-1ß) and N-terminal domain of gasdermin D (GSDMD-N) level. The oxidative stress and lipid peroxidation were also suppressed along with the up-regulation of the ferroptosis antagonism marker glutathione peroxidase-4 (GPX4) in Wed treatment group. Wed promoted the transcriptional activity and the selenium sensitivity of GPX4. Moreover, the protective effects of Wed in caerulein-stimulated pancreatic acinar cells were markedly abrogated by the down-regulation of GPX4. Collectively, our data suggest that pyroptosis and ferroptosis play crucial roles in AP. Wed mitigated AP and associated lung injury via GPX4 mediated suppression of pyroptosis and ferroptosis.

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.

Emodin Protects Against Acute Pancreatitis-Associated Lung Injury by Inhibiting NLPR3 Inflammasome Activation via Nrf2/HO-1 Signaling.

AIM: Lung injury is a common complication of acute pancreatitis (AP), which leads to the development of acute respiratory distress syndrome and causes high mortality. In the present study, we investigated the therapeutic effect of emodin on AP-induced lung injury and explored the molecular mechanisms involved. MATERIALS AND METHODS: Thirty male Sprague-Dawley rats were randomly divided into AP (n=24) and normal (n=6) groups. Rats in the AP group received a retrograde injection of 5% sodium taurocholate into the biliary-pancreatic duct and then randomly assigned to untreated, emodin, combined emodin and ML385, and dexamethasone (DEX) groups. Pancreatic and pulmonary injury was assessed using H&E staining. In in vitro study, rat alveolar epithelial cell line L2 cells were exposed to lipopolysaccharide and treated with emodin. Nrf2 siRNA pool was applied for the knockdown of Nrf2. The contents of the pro-inflammatory cytokines in the bronchoalveolar lavage fluid and lung were determined using enzyme-linked immunosorbent assay. The expressions of related mRNAs and proteins in the lung or L2 cells were detected using real-time polymerase chain reaction, Western blot, immunohistochemistry and immunofluorescence. KEY FINDINGS: Emodin administration alleviated pancreatic and pulmonary injury of rats with AP. Emodin administration suppressed the production of proinflammatory cytokines, downregulated NLRP3, ASC and caspase-1 expressions and inhibited NF-κB nuclear accumulation in the lung. In addition, Emodin increased Nrf2 nuclear translocation and upregulated HO-1 expression. Moreover, the anti-inflammatory effect of emodin was blocked by Nrf2 inhibitor ML385. CONCLUSION: Emodin effectively protects rats against AP-associated lung injury by inhibiting NLRP3 inflammasome activation via Nrf2/HO-1 signaling.