Serebrenik, Y. | Search bioRxiv

bioRxiv2024

Intracellular Protein Editing to Enable Incorporation of Non-Canonical Residues into Endogenous Proteins

The ability to study proteins in a cellular context is crucial to our understanding of biology. Here, we report a new technology for "intracellular protein editing", drawing from intein- mediated protein splicing, genetic code expansion, and endogenous protein tagging. This protein editing approach enables us to rapidly and site specifically install residues and chemical handles into a protein of interest. We demonstrate the power of this protein editing platform to edit cellular proteins, inserting epitope peptides, protein-specific sequences, and non-canonical amino acids (ncAAs). Importantly, we employ an endogenous tagging approach to apply our protein editing technology to endogenous proteins with minimal perturbation. We anticipate that the protein editing technology presented here will be applied to a diverse set of problems, enabling novel experiments in live mammalian cells and therefore provide unique biological insights. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=85 SRC="FIGDIR/small/602493v1_ufig1.gif" ALT="Figure 1"> View larger version (19K): org.highwire.dtl.DTLVardef@8957b8org.highwire.dtl.DTLVardef@de02beorg.highwire.dtl.DTLVardef@1863cb4org.highwire.dtl.DTLVardef@1fb8bc0_HPS_FORMAT_FIGEXP M_FIG C_FIG

biochemistry↗

bioRxiv2019

IRE1β negatively regulates IRE1α signaling in response to endoplasmic reticulum stress

IRE1{beta} is an ER stress sensor uniquely expressed in epithelial cells lining mucosal surfaces. Here, we show that intestinal epithelial cells expressing IRE1{beta} have an attenuated response to ER stress. IRE1{beta} assembles with and blocks activation of the closely related and most evolutionarily ancient stress-sensor IRE1 to suppress stress-induced xbp1 splicing, a key mediator of the unfolded protein response. In comparison, IRE1{beta} has weak xbp1 splicing activity, largely explained by a non-conserved amino acid in the kinase domain that impairs its phosphorylation and restricts oligomerization. This enables IRE1{beta} to act as a dominant negative suppressor of IRE1. The inhibitory effect is amplified in cells by disrupting an XBP1-dependent feedback loop regulating stress-induced expression of IRE1. Thus IRE1{beta} functions to negatively regulate IRE1 signaling, perhaps enabling intestinal epithelial cells to manage the response to chronic stress stimuli at the host-environment interface.

cell biology↗