Proteomics and model organism humanization to decode human genetics

Summary/Abstract While the human genome provides a parts list of >20,000 proteins, it is still largely unknown how these proteins assemble into ‘molecular machines’ to carry out their biological roles. This is important both for basic characterization of human genes and for understanding the mechanisms underlying most human genetic diseases, which often arise from defects in systems of proteins working together. We focus on the >9,000 human proteins shared across eukaryotes and dating to the last eukaryotic common ancestor. These ancient proteins carry out critical cellular processes, including DNA replication, repair, transcription, splicing, mitochondrial and ciliary processes, and trafficking, among others. They are disproportionately drivers of human disease, linked to a wide array of disorders, spanning cancers, birth defects, metabolic disorders, Parkinson disease, Huntington disease, amyotrophic lateral sclerosis, and more. Nearly 1,300 of these deeply conserved human proteins are still mostly uncharacterized, despite almost certainly having important cell roles. A fundamental question is how all of these proteins work together to support cell function. However, a key limitation remains the lack of large- scale data directly interrogating these proteins’ expression, interactions, and activation states. Current approaches to quantify the proteome are only beginning to survey the proteins in mammalian cells to any significant depth, and consistently suffer from low sensitivity and throughput. These limitations have slowed medical applications, e.g. biomarker discovery, where techniques including mass spectrometry and antibody arrays often lack sufficient sensitivity and quantification accuracy to be effective. We propose research in three broad areas: First, we propose a major effort to biochemically define the main human protein complexes, providing a mechanistic basis for interpreting diverse human genetics and diseases. We will focus on evolutionarily conserved human proteins due to these proteins’ critical importance to cellular function, leveraging studies in other species using a comparative proteomics approach. Second, we are developing surrogate functional assays for deeply conserved human proteins by systematically humanizing yeast cells, replacing each essential yeast gene in turn by its human version. The resulting strains serve as new physical reagents for studying human genes in a simplified organismal context, opening up simple high-throughput assays of human gene function, the impact of human genetic variation on gene function, the screening and repurposing of drugs, and the rapid determination of mechanisms of drug resistance. Finally, we aim to advance new proteomics technologies, single-molecule protein sequencing and shotgun electron microscopy, both of which enable new types of highly sensitive characterization of protein expression and physical organization relevant to many aspects of human cell biology and disease. Success of these aims will give new insights into basic human cell biology and biochemistry, laying the foundation for future attempts to intervene, chemically or genetically, with those macromolecules most critical to the functioning of cells.