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Synthetic 'killswitch' uncovers hidden world of cellular condensates

Researchers at the Max Planck Institute for Molecular Genetics have developed a novel synthetic micropeptide termed the "killswitch" to selectively immobilize proteins within cellular condensates, unveiling crucial connections between condensate microenvironments and their biological functions.

Biomolecular condensates are specialized regions inside cells, existing without membranes, where critical biochemical reactions occur. Their importance in health and disease is well established, including roles in cancer progression and viral infection.

Methods to precisely probe and manipulate condensates in living cells remain limited. Existing strategies lack specificity, either dissolving condensates indiscriminately or requiring artificial protein overexpression, which obscures the natural behavior of native cellular proteins.

In the study, "Probing condensate microenvironments with a micropeptide killswitch," in Nature, the authors engineered a short hydrophobic micropeptide capable of specifically targeting and immobilizing proteins within naturally occurring cellular condensates. By tagging this killswitch peptide to small antibody fragments, they directed it precisely to condensates labeled with fluorescent proteins.

The Max Planck team initially characterized this killswitch in human osteosarcoma cells and then expanded their analyses to a variety of endogenous condensates across different cell types.

Using antibody fragments to specifically recruit the killswitch into targeted condensates labeled with green fluorescent protein (GFP), they systematically manipulated condensates, such as nucleoli, nuclear speckles, and chromocentres, as well as pathological condensates, including those formed by cancer-driving fusion proteins and adenoviral proteins.

Employing human and mouse cell lines, the researchers tested the effects of the killswitch across these diverse condensate types.

Utilizing fluorescence recovery after photobleaching (FRAP), a microscopy-based method that measures how quickly proteins within condensates move and exchange, they observed a marked immobilization of targeted proteins. For example, nucleoli showed reduced dynamics and altered protein composition upon killswitch recruitment.

Further exploration using advanced mass spectrometry revealed specific proteins excluded from nucleoli following killswitch exposure, leading to functional consequences such as diminished ribosomal protein mobility and a separation from critical ribosomal RNA components.

Beyond fundamental cellular condensates, the team also tested disease-linked condensates. In cancer cells driven by fusion oncoproteins, misfit proteins formed through erroneous gene combinations.

The killswitch dramatically reduced condensate mobility and altered their internal composition. Notably, in a mouse model of acute myeloid leukemia driven by the fusion protein NUP98::KDM5A, the killswitch substantially impaired leukemic cell proliferation, hinting at the therapeutic potential of targeting condensate properties.

Versatility of the killswitch extended into viral pathology. Researchers targeted condensates formed by adenovirus protein 52K, critical for viral particle assembly. Here, the killswitch not only immobilized these viral condensates but also prevented necessary viral structural proteins from accumulating, and substantially reduced the virus by more than 90%.

Through their innovative killswitch micropeptide, the researchers have introduced a universally adaptable method for manipulating endogenous condensates.

With this approach in the toolkit, researchers will be able to significantly enhance our understanding of how condensate microenvironments influence cellular function and disease, and probe novel avenues for future therapeutic interventions in cancer and viral diseases.