“Deciphering the topology of molecular interaction networks is critical to understanding what goes wrong when cells become diseased.”
–Jeff Ranish, PhD, Associate Professor
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The Ranish group uses the comprehensive and high-throughput methods of systems biology to study the composition and architecture of large molecular complexes. These complexes, which can consist of multiple proteins, nucleic acids and small molecules, carry out many processes in cells, from protein synthesis to DNA replication and repair. By isolating the complexes and then using mass spectrometers to identify and quantify the proteins that make up these complexes, they can determine the composition of the complexes and how their compositions change during such processes as cell growth or differentiation. In addition, by employing chemical crosslinking reagents they can determine the architectural arrangement of the proteins within the complex. Both the architectural and the compositional information are critical for understanding how the complexes control cellular processes, which turn, is essential in using systems biology to transform medicine, energy production and environmental protection.
Cells use large molecular complexes to perform basic functions such as responding to the environment and replicating their DNA. Misregulation of these complexes can lead to devastating diseases such as cancer. To examine how these complexes work, the Ranish group studies their composition and architecture, producing essential information needed to adapt biological processes to serve human needs.
A particular focus of the research done by Jeff Ranish and his colleagues has been the complexes that transcribe gene sequences into messenger RNA (mRNA), which cells then use to make proteins. The group uses mass spectrometers to study the protein composition of the complexes and to determine how their composition changes in response to environmental and cellular events. For example, the group has shown that the complexes change composition during specific biological processes such as the differentiation of cells into mature red blood cells.
This basic biological research can have immediate applications. For example, Ranish and his colleagues recently discovered a new component of a transcription complex that is required for expression of most genes. When this molecule is mutated, it can cause a rare genetic disease known as tricothiodystrophy disorder (TTD), which is characterized by brittle hair and nails, scaly skin, photosensitivity, developmental defects, and other symptoms. The results of their studies suggest that patients with mutations in the molecule have defects in the formation of transcription complexes, which in turn leads to altered patterns of gene expression.
The Ranish group also is developing new mass spectrometry based approaches to systematically detect and quantify targeted proteins such as components of transcription complexes. By providing an in-depth understanding of how genes are turned on and off, the research will provide essential information in determining how cells function. Understanding these processes also will make it possible to reprogram the behavior of cells when the dysregulation of gene expression results in disease.
A particular innovation Ranish and his colleagues have developed is an approach to map the architecture of macromolecular complexes using mass spectrometry. They have developed reagents that form links between parts of a macromolecular complex that are in close proximity. Using powerful new software developed for this research, they use this information to piece together a model of the complex’s overall architecture. This information can then be used to develop strategies to control the activity of the complexes in desired ways.
Research Focus I
The first step in the expression of a gene is the formation of a preinitiation complex (PIC) at the site of the regulatory DNA that controls the gene’s activity. The formation of a PIC is itself a poorly understood and complex process that involves many proteins, only some of which have been identified. The Ranish group has shown that a protein active during the elongation phase of transcription, known as TFIIS, is also an essential player in the formation of a PIC. By creating truncated TFIIS proteins, they were able to show that part of the protein that binds to RNA polymerase II is needed for PIC assembly, though efficient assembly requires other parts of the protein.
Source: Kim B, Nesvizhskii AI, Rani PG, Hahn S, Aebersold R, Ranish JA. 2007. The transcription elongation factor TFIIS is a component of RNA polymerase II preinitiation complexes. Proceedings of the National Academy of Sciences 104:16068-73.
Research Focus II
Biomedical research requires protein detection technology that is not only sensitive and quantitative, but that can reproducibly measure any set of proteins in a biological system in a high throughput manner. The Ranish group recently developed a targeted proteomics platform termed index-ion triggered MS2 ion quantification (iMSTIQ) that allows reproducible and accurate peptide quantification in complex mixtures. Importantly, the method takes advantage of mass spectrometry instrumentation that provides data with high mass accuracy and resolution, in a high throughput manner. As such it provides an attractive tool to meet the demands of systems biology research and biomarker studies. The Ranish group is now employing this platform to systematically measure proteome changes during cellular differentiation and to probe the composition of gene regulatory complexes.
Source: Yan W, Luo J, Robinson M, Eng J, Aebersold R, Ranish J. 2011.
Index-ion triggered MS2 ion quantification: a novel proteomics approach for reproducible detection and quantification of targeted proteins in complex mixtures. Mol Cell Proteomics.