High Throughput Approaches
|Ubiquitin signaling networks||Sensitive assays for high content screening||Chemical biology and the receptorome|
The cellular protein landscape is dynamically regulated through changes in transcription and by the ubiquitin dependent degradation of specific proteins. In addition to its critical role in regulated protein degradation, the ubiquitin pathway has recently been shown to play a more complex role in regulating signal transduction, through controlling protein interactions and localization. The diversity of ubiquitin signaling outputs, and the perturbation of the ubiquitin proteasome system (UPS) in cancer underscore the importance of understanding key ubiquitin signaling events. However, a key challenge in the ubiquitin field has been to connect UPS enzymes with the substrates that they regulate. Our lab applies emerging genetic and proteomic technologies to systematically explore ubiquitin dependent signal transduction during cell cycle progression and in response to DNA damage. We are implementing and developing technologies that can assess global, proteome wide controlled by ubiquitination. Global Protein Stability Profiling (GPS) is a genetic platform that utilizes fluorescent reporters together with cell sorting to assess changes in protein stability. The GPS system employs a collection of more than 15,000 human open reading frames (ORFs) expressed from a fluorescent reporter construct to simultaneously assess changes in the stability of 15,000 human proteins. 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 and allow us to better understand global ubiquitin signaling networks regulated during cell growth, in response to stress and in during disease.
The image shows a novel merocyanine dye placed where a shift in the position of a charged amino acid affects a heteroatom on the dye, leading to a large increase in dye intensity. Proteins labeled with very bright, long wavelength dyes are used in both drug screening assays and to study protein conformation in vivo.
We have developed novel fluorescent dyes and labeling approaches for high content screening. The dyes are attached to proteins where their spectrum changes in response to protein phosphorylation, conformational changes or ligand binding. They have proven valuable in drug discovery, generating bright signals for sensitive screening assays. In addition, membrane permeable derivatives of the dyes are being attached to drug molecules to examine their metabolism and fate within living cells. Current work is focused on understanding the photophysical properties of dyes to build reporters of specific protein functions, reporting endogenous protein activity, and the ability to trace multiple different protein activities in the same assay or cell. In vivo labeling approaches are being harnessed to use dye-based biosensors in high content screening. Another side to this work is the generation of biosensors via high throughput screening, using libraries of engineered scaffolds. Library members binding to the activated conformation of targets are modified to generate biosensors of endogenous signaling proteins.
We have pioneered the approach of massively-parallel physical screening of the GPCR-ome. Our approach differs from conventional high-throughput (HTS) approaches in that we screen, in a parallel fashion, entire families of receptors simultaneously to discover molecular targets of biologically important molecules (peptides, drugs, natural products). This work is facilitated by the NIMH Psychoactive Drug Screening Program which is housed in the Roth lab.
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