Edited by Ursula Jakob Autophagy is of key importance for eliminating aggregated proteins during the maintenance of cellular proteostasis in response to endoplasmic reticulum (ER) stress. However, the upstream signaling that mediates autophagy activation in response to ER stress is incompletely understood. In this study, in vivo and in vitro approaches were utilized that include gainand loss-of-function assays and mouse livers and human cell lines with tunicamycin-induced pharmacological ER stress. We report that calreticulin, a quality control chaperone that binds to misfolded glycoproteins for refolding in the ER, is induced under ER stress. Calreticulin overexpression stimulated the formation of autophagosomes and increased autophagic flux. Interestingly, calreticulin was sufficient for attenuating ER stress in tunicamycin-or thapsigargin-treated HeLa cells, whereas lentivirus-mediated shRNA calreticulin knockdown exacerbated ER stress. Mechanistically, we noted that calreticulin induces autophagy by interacting with microtubule-associated protein 1A/1B-light chain 3 (LC3). Confocal microscopy revealed that the colocalization of calreticulin and LC3 at the autophagosome was enhanced under ER stress conditions. Importantly, a conserved LC3-interacting region was necessary for calreticulin-mediated stimulation of autophagy and for reducing ER stress. These findings indicate a calreticulin-based mechanism that couples ER stress to autophagy activation, which, in turn, attenuates cellular stress, likely by alleviating the formation of aberrantly folded proteins. Pharmacological or genetic approaches that activate calreticulin-autophagy signaling may have potential for managing ER stress and related cellular disorders. The endoplasmic reticulum (ER) 3 is a key site of protein synthesis and maturation, Ca 2ϩ storage, and lipid biogenesis (1).
Histone posttranslational modifications (PTMs) are vital epigenetic regulators in many fundamental cell signaling pathways and diverse biological processes. Histone lysine benzoylation is a recently identified epigenetic mark associated with active transcription; however, it remains to be explored. Herein, we first report the genetic encoding of benzoyllysine and fluorinated benzoyllysines into full-length histone proteins in a site-specific manner in live cells, based on our rationally designed synthetase and fine-integrated fluorine element into benzoyllysines. The incorporated unnatural amino acids integrating unique features were demonstrated as versatile probes for investigating histone benzoylation under biological environments, conferring multiplex signals such as 19F NMR spectra with chemical clarity and fluorescence signals for benzoylation. Moreover, the site specifically incorporated lysine benzoylation within native full-length histone proteins revealed distinct dynamics of debenzoylation in the presence of debenzoylase sirtuin 2 (SIRT2). Our developed strategy for genetic encoding of benzoyllysines offers a general and novel approach to gain insights into interactions of site-specific histone benzoylation modifications with interactomes and molecular mechanisms in physiological settings, which could not be accessible with fragment histone peptides. This versatile chemical tool enables a direct and new avenue to explore benzoylation, interactions, and histone epigenetics, which will provide broad utilities in chemical biology, protein science, and basic biology research.
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