Enzyme‐Instructed Assemblies Enable Mitochondria Localization of Histone H2B in Cancer Cells

It is known that a highly dynamic communication among subcellular organelles (e.g., cytosol, endoplasmic reticulum (ER), mitochondria, and nucleus) dictate cellular behaviors. But little information exists on how the inter-organelle crosstalk impacts cancer cells due to the lack of approaches that manipulate inter-organelle communication in cancer cells. We unexpectedly found that a negatively charged, enzyme cleavable peptide enables the trafficking of histone protein (H2B), a nuclear protein, to the mitochondria in cancer cells. The peptide, denoted as MitoFlag, interacts with the nuclear location sequence (NLS) of H2B to block it entering nucleus. A protease on the mitochondria cleaves the Flag from the complex of MitoFlag and H2B to form assemblies that retain H2B on the mitochondria and facilitate the H2B entering mitochondria. Molecular validation of MitoFlag shows that adding NLS, replacing aspartic acid residues by glutamic acid residues, or changing L-aspartic acid to D-aspartic residue abolishes the trafficking of H2B into mitochondria of HeLa cells. As the first example of enzyme-instructed self-assembly (EISA) of a synthetic peptide for trafficking endogenous proteins, this work provides insights for understanding and manipulating inter-organelle communication in cells.