Discovery and Characterization of Intracellularly Stable hnRNPA2B1 Specific Nanobodies for Live‐Cell Imaging and Targeted Protein Degradation

Abstract

AbstractBackgroundHeterogeneous nuclear ribonuclear proteins, hnRNPs, play a variety of roles in regulating transcriptional, and post‐transcriptional gene expression. A core member of the hnRNP family, hnRNPA2/B1 proteins are involved in the formation of membraneless‐organelles such as stress granules (SGs) under certain conditions. Mutations in hnRNPA2/B1 proteins are known to cause multisystem proteinopathy (MSP), a pleiotropic group of inherited disorders that cause neurodegeneration, myopathy, and bone disease. Mutations in hnRNPA2/B1 are also risk factors for the development of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). Despite growing evidence implicating hnRNPA2/B1 proteins in a multitude of diseases, our ability to study them in live cells is severely limited due to the lack of intracellularly stable hnRNPA2/B1 specific antibodies. Nanobodies (sdAbs) are antibody fragments derived from heavy‐chain‐only antibodies found in camelids and sharks. Owing to their small size (∼15 kDa) and unique biochemical properties, nanobodies emerged as promising alternatives to conventional‐antibody derived protein fragments. Nanobodies have superior stability that facilitate their intracellular expression. Full length antibodies and bulkier antibody‐derived fragments, such as single chain variable fragments (scFvs), are not amenable to intracellular expression, in part due to the reducing cytoplasmic environment that prevents the formation of disulfide bonds needed for their correct folding.MethodWe have developed a high‐throughput nanobody discovery platform from synthetic yeast surface display libraries by using antigen coated magnetics beads. Furthermore, we have developed generalizable and straightforward cellular assays to characterize nanobody specificity in live cells.ResultUsing above described platforms, we have identified novel hnRNPA2/B1 specific nanobodies and characterized their intracellular stability by expressing them in HEK293 cells. In addition to using nanobodies for studying hnRNPA2/B1 biology in live cells, we have also engineered a panel of modular E3 ubiquitin ligases targeting hnRNPA2/B1 for degradation. ∼30% reduction in hnRNPA2/B1 level was observed when targeted for degradation by using hnRNPA2B1 targeting E3 ubiquitin ligase.ConclusionIn summary, we have optimized factors affecting successful high‐throughput antibody discovery using yeast‐surface display antibody libraries. We also developed generalizable methods to characterize nanobody specificity in live cells. Platforms we have developed will help build tools to study protein aggregation and protein stability in live cells.