Maintaining protein homeostasis (proteostasis) requires the concerted action of sophisticated molecular pathways and assemblies within cells. Proteostasis regulation begins with ribosomes, which ensure the faithful translation of the genetic code by pairing each mRNA codon with the correct amino acid through a matching tRNA.

Events during these “formative minutes” can have long-lasting effects, since many nascent proteins start to fold and even interact with binding partners as they emerge from translating ribosomes. An unexpected key determinant of successful folding is the rate at which ribosomes elongate nascent chains. Elongation rates along each mRNA are not uniform and are dictated by several factors, chief among which is the interplay between synonymous codon usage and cellular tRNA supply. Protein folding is further assisted co- and post-translationally by molecular chaperones, while quality control systems continuously monitor proteome integrity and target aberrant molecules for destruction. Aging, persistent proteome damage, or genetic defects challenge this highly interconnected proteostasis network, and the resulting proteome imbalance is a hallmark of many human diseases.

In metazoans, the proteostasis network must be carefully tuned to varying demands in distinct cell types and throughout development. Cell type-specific differences in proteostasis regulation remain largely unexplored despite mounting evidence for their existence. For instance, mutations in ribosomal protein genes cause selective pathology in hematopoietic and neural crest cells, while defective tRNAs and chronic proteome damage often disrupt neuronal development and function. The expression of tRNAs, ribosomal proteins, chaperones, and quality control components is also remarkably heterogeneous among healthy cells and tissues. How this heterogeneity ensures normal cell physiology or how it accounts for cell type-specific sensitivity to proteome imbalance is poorly understood. To shed new light on these fundamental questions, we have established targeted differentiation of human induced pluripotent stem cells (hiPSCs) into a range of normal cellular states with distinct proteomes, as well as lineages selectively damaged by proteostasis defects in disease. This powerful model system enables us to dissect cell-specific proteostasis mechanisms and to study their contribution to normal development and disease.

To understand how metazoans achieve and maintain distinct cellular proteomes, we develop and employ a variety of quantitative genome-wide assays (e.g. tRNA sequencing , ChIP-Seq, ribosome profiling) and combine these with functional genomics screens. Novel mechanisms we identify with this discovery-based approach are dissected in molecular detail with targeted genetic, cellular, and biochemical assays.

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