Comprehensive analysis of WRN protein interaction network by Mass Spectrometry.

Abstract

Cells that have lost WRN function exhibit a shortened replicative lifespan, accumulation of chromosomal aberrations, and demonstrate sensitivity to a number of chemotherapeutic agents, including DNA Topoisomerase I inhibitor camptothecin (CPT). On organismal level, the lack of this protein results in the progeroid syndrome Werner Syndrome, which is characterized by increased incidence of cancers, cardiovascular disease, cataracts, and other age-associated pathologies. In this study we examined the network of proteins that associate with WRN protein, and then expanded this picture when the cells were challenged with CPT. We detail the profiling analysis used for unbiased detection of all interacting proteins using LC-MS/MS, followed by data analysis and selection of targets for follow-up. We then focus on the method development, complexities of data analysis, and application of selected reaction monitoring (SRM), a method for targeted MS. Using RNAi we demonstrate the power of this technique for relative protein quantitation of human proteins WRN and TP53, and a number of small, low-abundance seminal fluid proteins from D.melanogaster flies. SRM was applied for two studies of the WRN interactome: 1) to partially validate findings of the profiling screen after short-term treatment with CPT, and 2) to describe the dynamics of association of these proteins with WRN as a function of time, by expanding the treatment time course to 6 and 12 hours. We report on our description of WRN protein interaction complexes in unperturbed cells, and how these interaction complexes become reorganized in response to DNA damage. Our results demonstrate that the newly recruited proteins participate in chromatin remodeling, negative regulation of cell cycle progression, and double-strand break repair. We focus on 40 proteins to test their differential interaction with WRN after 1, 6, and 12 hours of treatment with CPT. We specifically focus on the functional importance of the chromatin-remodeling factors, and present the list of WRN interacting proteins that have emerged as high-priority targets for functional validation. Finally, we discuss how our work has expanded the understanding of the functional roles of WRN, and how targeted, hypothesis-driven application of mass spectrometry can be used to answer complex questions about protein interaction.