Department of Pharmacology
Ph.D., Biochemistry and Molecular Genetics
University of Virginia
- cell cycle, mitosis, protein stability, ubiquitin, cancer, genetics, cell biology
Systematic examination of proteome organization
Human cells have the ability to encode more than twenty thousand unique proteins. The protein landscape, or “proteome” (which proteins and the respective levels of each), can be vastly different between different cell types. Importantly, cells can dynamically reorganize their proteome in response to environmental changes and stress, during development, throughout the cell cycle and during cancer initiation and progression. The proteome is determined primarily through two distinct mechanisms: regulated transcription of gene expression and targeted degradation of specific proteins. While there are robust methods that analyze global changes in transcription, limited tools are available to assess global changes in protein degradation, or stability. Our lab is interested in applying emerging genetic and proteomic techniques to gain a systems level understanding of global changes in protein stability. Moreover, since cancer is primarily a disease of cell cycle de-regulation, we are focused on identifying the regulatory circuits controlling proteins that are transcribed and degraded throughout the cell cycle. In addition, DNA damaging agents and mitotic inhibitors are the two most commonly used clinical anti-cancer agents and we are therefore also focused on how these compounds affect proteome reorganization.
To address these challenges in a systematic way, we are implementing and developing technologies that can assess global, proteome wide changes in protein stability. Global Protein Stability Profiling (GPS) is a genetic platform that utilizes fluorescent reporters together with cell sorting to assess changes in protein stability (see reference 1 below). The GPS system employs a collection of more than 15,000 human ORFs (based on the human ORFeome collection) expressed from a retroviral reporter construct that encodes red fluorescent protein (DsRed) and green fluorescent protein (GFP) fused to the protein of interest. Importantly, GPS can simultaneously assess changes in the stability of these 15,000 proteins, and effectively analyzes low abundance proteins often missed by proteomic techniques. As a complement to GPS, we utilize a proteomic approach termed QUAINT (Quantitative Ubiquitylation Interrogation). QUAINT is a mass spectrometry based platform that quantitatively measures changes in protein ubiquitylation for endogenous proteins. Together, these emerging technologies will provide a deep snap shot in the regulated proteome.
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Click above for PubMed publications.
- Emanuele MJ, Elia EH, Xu Q, Thoma CR, Izhar L, Guo A, Rush J, Hsu PW, Yen HS, Elledge SJ. Global Identification of Modular Cullin-Ring Ligase Substrates. Cell. 2011 Oct 14;147(2):459-74. Epub 2011 Sep 29.
- Emanuele MJ, Ciccia A, Elia AE, Elledge SJ. Proliferating cell nuclear antigen (PCNA)-associated KIAA0101/PAF15 protein is a cell cycle-regulated anaphase-promoting complex/cyclosome substrate. PNAS 2011. 108 (24) 9845-9850;
- Luo J, Emanuele MJ, Li D, Creighton CJ, Schlabach MR, Westbrook TF, Wong K, Elledge SJ. A genome-wide RNAi screen identifies multiple synthetic lethal interactions with the Ras oncogene. Cell. 2009 May 29; 137(5). 835-48.
- Emanuele MJ, Lan W, Jwa M, Miller SA, Chan, CSM, Stukenberg PT. Aurora B kinase and Protein Phosphatase 1 have opposing roles in modulating kinetochore assembly. J Cell Biol. 2008 Apr 21;181(2):241-54.
- Emanuele MJ and Stukenberg PT. Xenopus Cep57 is a novel kinetochore component involved in microtubule attachment. Cell. 2007 Sep 7;130(5):893-905.
- Emanuele MJ*, McCleland ML*, Satinover DL, Stukenberg PT. Measuring the stoichiometry and physical interactions between components elucidates the architecture of the vertebrate kinetochore. Mol Biol Cell. 2005 Oct;16(10):4882-92. (*authors contributed equally to this work)
Lineberger Comprehensive Cancer Center