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Genomic Analysis of Protein Function
Summary: Stanley Fields analyzes the function of proteins from the yeast Saccharomyces cerevisiae on a genome-wide basis and uses this yeast to develop assays that can be applied to proteins from any organism.
The past decade has seen a profusion of whole-genome sequences, with total DNA sequence accumulation in GenBank now more than 100 billion bases. Genome sequences have led to the prediction of large complements of proteins, ranging from a few thousand in bacterial species to more than 20,000 for humans and other mammalian species. However, the determination of protein function remains a difficult task, given the tremendous range of biochemical activities that proteins display, the diverse modifications that a protein can undergo during its lifetime, the multiplicity of proteins potentially encoded by a single gene, and the use of proteins for more than a single function.
Our laboratory is interested in developing technologies, especially those to analyze protein function. For many of our efforts, we use the unicellular eukaryote Saccharomyces cerevisiae (baker's yeast) as the host organism for carrying out protein assays. Yeast—the first eukaryote to be sequenced—has a relatively small number of genes, is highly tractable for experimentation, and has been used to derive numerous sets of reagents and high-throughput data. As a consequence, the set of yeast proteins is particularly advantageous for testing new technologies. In addition, yeast is a convenient host to express proteins from many other organisms, and we have taken advantage of this property to analyze several sets of such heterologous proteins.
We have also used S. cerevisiae for the analysis of proteins relevant to human disease. Past studies have focused on a human polyglutamine-containing protein implicated in neurodegenerative disease, the human Toll-like receptors that mediate innate immunity, the proteins of the malaria parasite Plasmodium falciparum, and yeast proteins that play a role in aging.
Protein Interactions
We developed an array format in which nearly all of the predicted open reading frames of S. cerevisiae were generated as fusions with the activation domain of the yeast Gal4 transcription factor. An automated procedure is used to screen the array via the two-hybrid method. More than 1,000 proteins have been screened so far, with the resultant identification of thousands of putative interactions. With Prolexys Pharmaceuticals Inc. (Salt Lake City), we carried out a high-throughput two-hybrid analysis of P. falciparum, the parasite responsible for the most virulent form of malaria. With Min-Hao Kuo (Michigan State University), we modified the two-hybrid method to detect interactions that depend on a post-translational modification.
With Jing Huang (University of California, Los Angeles) and Invitrogen Inc. (Branford, Connecticut), we developed a pooling strategy that can dramatically decrease the effort required to generate large-scale datasets. With Invitrogen, we used protein microarray technology to generate a protein interaction map for 12 of the 13 WW domains of S. cerevisiae. We observed nearly 600 interactions between these 12 domains and ~200 proteins.
Protein Display Technology
The increased availability of gene sequences requires technologies to enable screens of the encoded polypeptides for diverse binding and catalytic activities. We are exploring technologies to couple DNA fragments, the mRNA transcribed in vitro from this DNA, and the protein translated in vitro from this RNA to provide the basis for activity screens and binding selections. This technology could be applied to complex mixtures of DNA, such as those present in the gut microbiota.
Substrate-Enzyme Relationships in Ubiquitination
Ubiquitin is a highly conserved protein whose attachment to a target protein can alter its fate. The E1 ubiquitin-activating enzyme, E2 ubiquitin-conjugating enzymes, and E3 ubiquitin ligases act in concert to covalently attach ubiquitin to proteins. There are 35 E3 ligases in S. cerevisiae and nearly 1,000 putative ones in humans, many of which, such as the breast cancer–specific tumor-suppressor BRCA1 and early-onset Parkinson's disease protein Parkin, are important in disease. We are using several approaches to identify on a genome-wide basis the specific protein substrates of individual E3 enzymes.
Metabolites in Yeast
Metabolism encompasses all the processes by which a cell generates energy and other essential molecules from nutrients. These pathways rely on hundreds of genes and involve thousands of small molecules. Using fluorescent reagents and capillary electrophoresis, we are profiling metabolites in S. cerevisiae—which shares all major metabolic pathways with humans—to determine the effects of genetic and environmental stimulation on metabolism. We hope to apply these data to understanding human mutations and polymorphisms affecting metabolic pathways, yielding insights into the biology of metabolic processes in human health and disease.
Protein-DNA Interactions
We are addressing the question of what sites in the genome are bound by DNA-binding proteins, one of the central issues in gene regulation. In collaboration with the laboratories of John Stamatoyannopoulos and William Noble (University of Washington), we developed a method, termed "digital genomic footprinting," to identify these sites on a genome-wide basis. The method is based on the classic treatment of chromatin with DNaseI but identifies cleavage sites genome-wide by high-throughput DNA sequencing. Using yeast as a test case, we identified thousands of "footprints"—short stretches of sequence that are protected against DNaseI digestion—and correlated them with known transcription factor motifs, gene regulation, and nucleosome patterns. This approach has the potential to delineate the transcriptional regulatory framework of any organism with an available genome sequence.
Genome-wide RNA Analysis
RNAs are implicated in an increasing number of regulatory activities. We are working on a set of projects to characterize yeast RNAs, RNA secondary structure, and RNA-protein interactions on a genome-wide basis. In one project, we seek to identify base-paired or protein-bound RNA sites by treating RNA with an RNase and copying surviving RNA fragments to cDNA for high-throughput sequencing. In a second project, we identify specific classes of RNAs, such as those produced by catalytic RNA cleavage and those that contain 2',3'-cyclic phosphates. Finally, we are combining a transcription run-on assay with sequencing to quantify transcription rates on a genome-wide basis; this approach should delineate rapid changes in the mRNA population upon environmental perturbations.
Chromosome Structure
Evidence from work in several organisms indicates that specific interchromosomal interactions occur and can affect gene expression and other processes. We are analyzing yeast chromosomes to identify specific contacts between genomic loci. In one approach, we examine a single site in the yeast genome in an effort to characterize changes in chromosome structure upon pheromone treatment of yeast. In another, we seek to identify links between yeast chromosomes on a genome-wide basis.
Yeast Aging
S. cerevisiae is a useful organism for studying factors that determine cellular longevity. The aging of mitotically active cells in higher eukaryotes can be modeled by the replicative life span of yeast mother cells, whereas the aging of postmitotic cells resembles the chronological survival of quiescent yeast during stationary phase. We used high-throughput technologies to identify and characterize genes that modify both aspects of the cellular life span. With Brian Kennedy (University of Washington), we determined replicative life span for a significant fraction of the strains in the S. cerevisiae deletion collection and chronological life span for all of the strains. Among the genes identified in both the replicative and chronological assays, several are known components of the TOR signaling pathway. The TOR pathway may be a primary conduit through which excess nutrient availability promotes aging in eukaryotic cells.
Some of this research was supported by grants from the National Institutes of Health.
Last updated: October 14, 2008
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