Abstract
Global change in protein turnover (protein degradome) constitutes a central part of cellular responses to intrinsic or extrinsic stimuli. However, profiling protein degradome remains technically challenging. Recently, inhibition of the proteasome, e.g., by using bortezomib (BTZ), has emerged as a major chemotherapeutic strategy for treating multiple myeloma and other human malignancies, but systematic understanding of the mechanisms for BTZ drug action and tumor drug resistance is yet to be achieved. Here we developed and applied a dual-fluorescence-based Protein Turnover Assay (ProTA) to quantitatively profile global changes in human protein degradome upon BTZ-induced proteasomal inhibition. ProTA and subsequent network analyses delineate potential molecular basis for BTZ action and tumor drug resistance in BTZ chemotherapy. Finally, combined use of BTZ with drugs targeting the ProTA-identified key genes or pathways in BTZ action reduced BTZ resistance in multiple myeloma cells. Remarkably, BTZ stabilizes proteasome subunit PSMC1 and proteasome assembly factor PSMD10, suggesting a previously under-appreciated mechanism for regulating proteasome homeostasis. Therefore, ProTA is a novel tool for profiling human protein degradome to elucidate potential mechanisms of drug action and resistance, which might facilitate therapeutic development targeting proteostasis to treat human disorders.
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Acknowledgements
We are grateful to Dr W Bian and her team in Cell Biology Core facility of SIBCB for their excellent support in flow cytometry analysis and cell sorting, and Drs L Lin, Y Xia and H Xiao of National Center for Microarrays for their help in microarray analysis. We are particularly grateful to Dr Hsueh-Chi Yen at Institute of Mol Biol, Academia Sinica, and Dr Ivan Dikic of Goethe University for advice, and Drs Dangsheng Li, Lijian Hui and Jiarui Wu of SIBCB, and Dr Shirley Diamond of California Institute of Technology for helpful discussion. We also thank Dr Ya-lan Wu and all other members of the Hu lab for support. This work was supported by the National Natural Science Foundation of China (31270828, 31070678, 81170491, 81072070 and 81470360), the 100 Talents award from CAS to RH and the Ministry of Science and Technology, China (2010CB912100, 2012CB910800 and 2013CB910900 to RH, and 2011CB915501 to KR). RH was also supported by a Sanofi-aventis SIBS Young Investigator award and funding from the Cancer Center of Xuhui Central Hospital (CCR2012003), Shanghai Institute of Neurosciences (SKLN-201206) and the Instrument Developing Project of the Chinese Academy of Sciences (YZ201339).
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Supplementary information
Supplementary information, Figure S1
Immunoblotting analysis confirmed bortezomib induced changes in stability of individual fusion proteins. (PDF 608 kb)
Supplementary information, Figure S2
Immunoblotting analysis confirmed bortezomib-induced changes in stability of endogenous proteins. (PDF 542 kb)
Supplementary information, Figure S3
Categorization of bortezomib-induced changes in global protein stability. (PDF 478 kb)
Supplementary information, Figure S4
Protein-protein interaction (PPI) network analysis of bortezomib-induced changes in protein stability. (PDF 392 kb)
Supplementary information, Figure S5
Characterization of BTZ-sensitive cells (CZ-1) or BTZ-resistant cells (CZ-1/R) from a multiple myeloma (MM) patient48. (PDF 375 kb)
Supplementary information, Figure S6
(A) The stability of PSMC1 and PSMD10 were assayed by cycloheximide (CHX) chase in CZ-1 cells. (PDF 871 kb)
Supplementary information, Figure S7
Categorization of bortezomib-induced changes in gene transcriptions. (PDF 427 kb)
Supplementary information, Figure S8
Protein-protein interaction (PPI) network analysis of bortezomib-induced changes in gene transcriptions. (PDF 258 kb)
Supplementary information, Figure S9
Chemical-protein interaction (CPI) network analysis of bortezomib induced changes in protein stability. (PDF 234 kb)
Supplementary information, Figure S10
Combined chemotherapy using BTZ and drugs that targeted the ProTA identified key genes (PDF 302 kb)
Supplementary information, Figure S11
C75, 17-AAG and BTZ exhibit synergistic, cytotoxic effects on the MM cell line CZ-1 (PDF 292 kb)
Supplementary information, Table S1
Annotations for the genes whose symbols were frequently used in this work (XLSX 19 kb)
Supplementary information, Table S2
A complete list of the ProTA-identified protein stability profiles in 293FT cells treated with or without bortezomib (XLSX 2287 kb)
Supplementary information, Data S1
Materials and Methods (PDF 265 kb)
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Yu, T., Tao, Y., Yang, M. et al. Profiling human protein degradome delineates cellular responses to proteasomal inhibition and reveals a feedback mechanism in regulating proteasome homeostasis. Cell Res 24, 1214–1230 (2014). https://doi.org/10.1038/cr.2014.122
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DOI: https://doi.org/10.1038/cr.2014.122
