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.
Covalent chemical reactions to modify aggregated proteins are rare.Here,wereported covalent Michael addition can generally occur upon protein aggregation. Suchr eactivity was initially discovered by ab ioinspired fluorescent colorswitch 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 noncovalently bound to misfolded proteins.S upported by the biochemical and mass spectrometry results,t he 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 chemicalt unability,r esulting in different fluorescence color-switch responses.O ur work may offer an ew avenue to explore other chemical reactions upon protein aggregation and design covalent probes for imaging,c hemical proteomics,and therapeutic purposes.
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