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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., (© 2021 Wiley-VCH GmbH.)

Protein phase separation process involving protein unfolding, misfolding, condensation and aggregation etc. has been associated with numerous human degenerative diseases. The complexity in protein conformational transitions results in multi-step and multi-species biochemical pathways upon protein phase separation. Recent progresses in designing novel fluorescent probes have unraveled the enriched details of phase separated proteins and provided mechanistic insights towards disease pathology. In this review, we summarized the design and characterizations of fluorescent probes that selectively illuminated proteins at different phase separated states with a focus on aggregation-induced emission probes, fluorescent molecular rotors, and solvatochromic fluorophores. Inspired by these pioneering works, a design blueprint was proposed to further develop fluorescent probes that can potentially shed light on the unresolved protein phase separated states in the future., (© 2021 Wiley-VCH GmbH.)

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.

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Background: Early exercise after stroke promoted angiogenesis and increased microvessles density. However, whether these newly formatted vessels indeed give rise to functional vascular and improve the cerebral blood flow (CBF) in impaired brain region is still unclear. The present study aimed to determine the effect of early exercise on angiogenesis and CBF in ischemic region.Methods: Adult male Sprague Dawley rats were subjected to 90 min middle cerebral artery occlusion(MCAO)and randomly divided into early exercise and non-exercised control group 24 h later. Two weeks later, CBF in ischemic region was determined by laser speckle flowmetry(LSF). Meantime, micro vessels density, the expression of tie-2, total Akt and phosphorylated Akt (p-Akt), and infarct volume were detected with immunohistochemistry, 2,3,5 triphenyltetrazolium chloride (TTC) staining and western blotting respectively. The function was evaluated by seven point's method.Results: Our results showed that CBF, vessel density and expression of Tie-2, p-Akt in ischemic region were higher in early exercise group compared with those in non-exercise group. Consistent with these results, rats in early exercise group had a significantly reduced infarct volume and better functional outcomes than those in non-exercise group.Conclusions: Our results indicated that early exercise after MCAO improved the CBF in ischemic region, reduced infarct volume and promoted the functional outcomes, the underlying mechanism was correlated with angiogenesis in the ischemic cortex. [ABSTRACT FROM AUTHOR]