Subcellular proteomics reveals drug-induced compartment-specific protein abundance changes and translocation.

Abstract

Eukaryotic cells, with their complex organelles and internal membrane systems, perform a variety of cellular processes. A critical factor in these processes is the subcellular localization of proteins. Current proteomics research on drug mechanisms primarily focuses on whole-cell studies, which are inadequate for effectively analyzing the dynamic molecular networks and mechanisms between subcellular compartments. This study utilized subcellular fraction proteomics after treating cells with 30 clinically approved drugs and revealed that different drugs induced markedly different changes in subcellular fraction protein abundance. Among the 30 drugs, Panobinostat induced the most pronounced changes in subcellular fraction proteins, with the highest downregulation rate observed in mitochondrial proteins at 32.5%, while nuclear proteins changed least, exhibiting an upregulation rate of 18.6%. Panobinostat downregulated the intrinsic apoptotic signaling pathway in the cytosol and nucleus, while upregulating it in mitochondria. The mTOR signaling pathway was significantly enriched among upregulated cytosolic proteins. Drugs sharing the same target exhibited markedly different effects on the same subcellular fraction, a divergence that was especially pronounced in the nucleus. Afatinib, an EGFR inhibitor, significantly downregulated 27.8% of nuclear proteins, whereas Osimertinib, another EGFR inhibitor, significantly downregulated only 0.4% of nuclear proteins. At the same time, both Afatinib and Osimertinib induced significant downregulation of proteins of multiple mitochondrial biological processes, consistent with mitochondrial dysfunction. Further analyses revealed candidate protein translocation events between subcellular fractions. Specifically, nucleophosmin (NPM1) translocated from the nucleus to the cytosol in response to paclitaxel, a change that was not detectable at the whole-cell proteomic level. The study also demonstrated marked differences across subcellular fractions in the proteins that co-vary in abundance with the same protein complex. In the cytosol, the CCT complex correlated strongly and positively with DYNC1H1, whereas in the nucleus it correlated strongly and positively with COPA and EIF4B. These correlations describe compartment-specific co-abundance patterns rather than physical interactions. As these observations are derived from a single cell model, they should be regarded as hypothesis-generating and require validation in additional cell lines. SIGNIFICANCE: The subcellular localization of proteins is fundamental to eukaryotic cell function, yet drug mechanism studies still rely predominantly on whole-cell proteomics, which obscures compartment-specific responses and communication between subcellular fractions. Here we profiled the nuclear, mitochondrial and cytosolic proteomes of HEK293T cells treated with 30 clinically approved drugs. Drug effects were highly compartment-selective, and compounds sharing the same molecular target produced divergent proteomic signatures in different fractions, revealing a layer of specificity that whole-cell analyses conceal. We also identified candidate protein translocation events between fractions, including an NPM1 redistribution from nucleus to cytosol that was not detectable at the whole-cell level, and found that co-abundance correlation networks of the same protein complex differ among fractions. These observations position subcellular proteomics as a framework for resolving drug action at the compartment level and for generating hypotheses about compartment-specific drug targets and translocation-based pharmacodynamic markers.

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