Biology subjects

Serebrenik, Y. V.

Publications and source records attributed to Serebrenik, Y. V..

3 recordsLinked to original sources

bioRxiv2023

Pooled tagging and hydrophobic targeting of endogenous proteins for unbiased mapping of unfolded protein responses

System-level understanding of proteome organization and function requires methods for direct visualization and manipulation of proteins at scale. We developed an approach enabled by high-throughput gene tagging for the generation and analysis of complex cell pools with endogenously tagged proteins. Proteins are tagged with HaloTag to enable visualization or direct perturbation. Fluorescent labeling followed by in situ sequencing and deep learning-based image analysis identifies the localization pattern of each tag, providing a birds-eye-view of cellular organization. Next, we use a hydrophobic HaloTag ligand to misfold tagged proteins, inducing spatially restricted proteotoxic stress that is read out by single cell RNA sequencing. By integrating optical and perturbation data, we map compartment-specific responses to protein misfolding, revealing inter-compartment organization and direct crosstalk, and assigning proteostasis functions to uncharacterized genes. Altogether, we present a powerful and efficient method for large-scale studies of proteome dynamics, function, and homeostasis.

cell biology↗

bioRxiv2023

Pooled endogenous protein tagging and recruitment for scalable discovery of effectors for induced proximity therapeutics

The field of induced proximity therapeutics is in its ascendancy but is limited by a lack of scalable tools to systematically explore effector-target protein pairs in an unbiased manner. Here, we combined Scalable POoled Targeting with a LIgandable Tag at Endogenous Sites (SPOTLITES) for the high-throughput tagging of endogenous proteins, with generic small molecule-based protein recruitment to screen for novel proximity-based effectors. We apply this methodology in two orthogonal screens for targeted protein degradation: the first using fluorescence to monitor target protein levels directly, and the second using a cellular growth phenotype that depends on the degradation of an essential protein. Our screens revealed a multitude of potential new effector proteins for degradation and converged on members of the CTLH complex which we demonstrate potently induce degradation. Altogether, we introduce a platform for pooled induction of endogenous protein-protein interactions that can be used to expand our toolset of effector proteins for targeted protein degradation and other forms of induced proximity.

cell biology↗

bioRxiv2018

Scalable tagging of endogenous genes by homology-independent intron targeting

Genome editing tools have simplified the generation of knock-in gene fusions, yet the requirement for gene-specific homology directed repair (HDR) templates still hinders the scalability of most approaches. Here, we combine intron-based protein trapping with homology independent repair-based editing and demonstrate precise and efficient gene tagging that can be easily scaled due to use of a generic donor. As editing is done in introns, this approach tolerates mutations in the unedited allele, disruptive indels, and allows for flexible donor and sgRNA design.

cell biology↗