February 24
2011
Biodesign Auditorium
727 E. Tyler St. Tempe
AZ 85287
One of the most compelling steps in the post-genomic era is learning the functional roles for all proteins. We have developed the FLEXGene Repository (for Full-Length Expression-ready), which comprises over 8000 full length clones for human genes, as well as complete genomes collections for several microorganisms enabling the high-throughput (HT) screening of protein function for the entire set (or any customized subset) of genes using any method of in vitro or in vivo expression.
The clones from the repository can be rapidly incorporated in HT biological experimentation. Using HT retroviral methods, proteins capable of inducing cancer-like behavior and conferring drug resistance can be tested. We derived a series of sub-cloned MCF7 cell lines that were either highly sensitive or naturally resistant to tamoxifen and studied the factors that lead to drug resistance. Gene expression studies revealed a signature of 67 genes that differentially respond to tamoxifen in sensitive vs. resistant subclones that also predicts disease-free survival in tamoxifen treated patients. High-throughput cell-based screens, in which >500 human kinases were independently ectopically expressed, identified 31 kinases that conferred drug resistance on sensitive cells. One of these, HSPB8, was also in the expression signature and, by itself, predicts poor clinical outcome in patients. Further studies revealed that HSPB8 protects MCF7 cells from tamoxifen and blocks autophagy. Moreover, silencing HSBP8 induces autophagy and causes cell death.
We developed a novel form of protein microarray, called nucleic acid programmable protein array (NAPPA). In lieu of producing and printing purified proteins, this method substitutes the printing of cDNAs encoding the proteins. Thus, the resulting array is a DNA array that can be converted into a protein array by adding cell free protein synthesis machinery. This obviates the need to purify proteins, produces human proteins in a mammalian milieu, and avoids concerns about protein stability on the array because the proteins are made just-in-time for assay. Moreover, the method displays a broad variety of proteins, insensitive to protein class or size with a high yield of protein per feature while maintaining a narrow range of protein yield from protein to protein.
NAPPA arrays can be used to study protein-protein interactions, protein-drug interactions, search for enzyme substrates, and as tools to search for disease biomarkers. In particular, recent experiments have focused on using these protein microarrays to search for autoantibody responses in cancer patients. Several bona fide autoantibody responses, such as responses to p53, have been detected, and a pilot study of responses to 7500 full length human proteins in 50 breast cancer patients and 50 controls has identified over 700 candidate proteins with more frequent responses in patients. These experiments show promise in finding antibody responses that appear in only cancer patients.
