Abstract
We report the orthologous screening, engineering, and optimization of amino acid conversion enzymes for cell-specific proteomic labeling. Intracellular endoplasmic-reticulum-anchored Mycobacterium tuberculosis diaminopimelate decarboxylase (DDC(M.tub-KDEL)) confers cell-specific meso-2,6-diaminopimelate-dependent proliferation to multiple eukaryotic cell types. Optimized lysine racemase (Lyr(M37-KDEL)) supports D-lysine specific proliferation and efficient cell-specific isotopic labeling. When ectopically expressed in discrete cell types, these enzymes confer 90% cell-specific isotopic labeling efficiency after 10 days of co-culture. Moreover, DDC(M.tub-KDEL) and Lyr(M37-KDEL) facilitate equally high cell-specific labeling fidelity without daily media exchange. Consequently, the reported novel enzyme pairing can be used to study cell-specific signaling in uninterrupted, continuous co-cultures. Demonstrating the importance of increased labeling stability for addressing novel biological questions, we compare the cell-specific phosphoproteome of fibroblasts in direct co-culture with epithelial tumor cells in both interrupted (daily media exchange) and continuous (no media exchange) co-cultures. This analysis identified multiple cell-specific phosphorylation sites specifically regulated in the continuous co-culture. Given their applicability to multiple cell types, continuous co-culture labeling fidelity, and suitability for long-term cell-cell phospho-signaling experiments, we propose DDC(M.tub-KDEL) and Lyr(M37-KDEL) as excellent enzymes for cell-specific labeling with amino acid precursors.
Highlights
From the ‡Division of Cancer Biology, The Institute of Cancer Research, 237 Fulham Road, London, SW3 6JB, UK; §Department of Biological Engineering, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139
Only one cell line has been successfully established with DAP-dependent growth, and the authors note that several other cell lines do not proliferate efficiently with DDCA.tha
There is currently no empirical evidence that DDCA.tha is retained within eukaryotic cells, which could lead to compromised labeling fidelity in co-culture
Summary
Cell-specific Labeling Enzymes for Analysis of Cell–Cell Communication in Continuous Co-culture*□S. This analysis identified multiple cell-specific phosphorylation sites regulated in the continuous co-culture Given their applicability to multiple cell types, continuous co-culture labeling fidelity, and suitability for long-term cell– cell phospho-signaling experiments, we propose DDCM.tub-KDEL and LyrM37-KDEL as excellent enzymes for cell-specific labeling with amino acid precursors. As L-lysine and L-arginine are metabolized by all cell types in a co-culture, SILAC technology is not suitable for investigating long-term cell-specific proteomic changes in proliferating co-cultures To address this limitation, Gauthier et al recently reported an alternative cell-specific isotopic labeling technology [4]. Given the broad potential application of cell-specific labeling with amino acid precursors, we sought to develop an optimal enzyme pairing capable of conferring high-fidelity, cell-specific, isotopic labeling to multiple cell types. We report the screening, engineering, and characterization of optimized DDC and Lyr enzymes for use in cell-specific labeling with amino acid precursors
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