Biology subjects

Lee, K. A.

Publications and source records attributed to Lee, K. A..

4 recordsLinked to original sources

bioRxiv2024

Transfer RNA acetylation regulates in vivo mammalian stress signaling

Transfer RNA (tRNA) modifications are crucial for protein synthesis, but their position-specific physiological roles remain poorly understood. Here we investigate the impact of N4-acetylcytidine (ac4C), a highly conserved tRNA modification, using a Thumpd1 knockout mouse model. We find that loss of Thumpd1-dependent tRNA acetylation leads to reduced levels of tRNALeu, increased ribosome stalling, and activation of eIF2 phosphorylation. Thumpd1 knockout mice exhibit growth defects and sterility. Remarkably, concurrent knockout of Thumpd1 and the stress-sensing kinase Gcn2 causes penetrant postnatal lethality, indicating a critical genetic interaction. Our findings demonstrate that a modification restricted to a single position within type II cytosolic tRNAs can regulate ribosome-mediated stress signaling in mammalian organisms, with implications for our understanding of translation control as well as therapeutic interventions.

developmental biology↗

bioRxiv2021

Novel antibodies for the simple and efficient enrichment of native O-GlcNAc modified peptides

Antibodies against post-translational modifications (PTMs) such as lysine acetylation, ubiquitin remnants, or phosphotyrosine have resulted in significant advances in our understanding of the fundamental roles of PTMs in biology. However, the roles of a number of PTMs remain largely unexplored due to the lack of robust enrichment reagents. The addition of N-acetylglucosamine to serine and threonine residues (O-GlcNAc) by the O-GlcNAc transferase (OGT) is a PTM implicated in numerous biological processes and disease states but with limited techniques for its study. Here, we evaluate a new mixture of anti-O-GlcNAc monoclonal antibodies for the immunoprecipitation of native O-GlcNAcylated peptides from cells and tissues. The anti-O-GlcNAc antibodies display good sensitivity and high specificity toward O-GlcNAc-modified peptides, and do not recognize O-GalNAc or GlcNAc in extended glycans. Applying this antibody-based enrichment strategy to synaptosomes from mouse brain tissue samples, we identified over 1,300 unique O-GlcNAc-modified peptides and over 1,000 sites using just a fraction of sample preparation and instrument time required in other landmark investigations of O-GlcNAcylation. Our rapid and robust method greatly simplifies the analysis of O-GlcNAc signaling and will help to elucidate the role of this challenging PTM in health and disease.

biochemistry↗

bioRxiv2021

Automating UbiFast for High-throughput and Multiplexed Ubiquitin Enrichment

Robust methods for deep-scale enrichment and site-specific identification of ubiquitylation sites is necessary for characterizing the myriad roles of protein ubiquitylation. To this end we previously developed UbiFast, a sensitive method for highly multiplexed ubiquitylation profiling where K-{varepsilon}-GG peptides are enriched with anti-K-{varepsilon}-GG antibody and labeled on-antibody with isobaric labeling reagents for sample multiplexing. Here, we present robotic automation of the UbiFast method using a magnetic bead-conjugated K-{varepsilon}-GG antibody (mK-{varepsilon}-GG) and a magnetic particle processor. We report the identification of [~]20,000 ubiquitylation sites from a TMT10-plex with 500 g input per sample processed in [~]2 hours. Automation of the UbiFast method greatly increased the number of identified and quantified ubiquitylation sites, improved reproducibility and significantly reduced processing time. The workflow enables processing of up to 96 samples in a single day making it suitable to study ubiquitylation in large sample sets. Here we demonstrate the applicability of this method to profile small amounts of tissue using breast cancer patient-derived xenograft (PDX) tissue samples.

biochemistry↗

bioRxiv2017

Restriction of dietary protein leads to conditioned protein preference and elevated palatability of protein-containing food in rats.

The mechanisms by which intake of dietary protein is regulated are poorly understood despite their potential involvement in determining food choice and appetite. In particular, it is unclear whether protein deficiency results in a specific appetite for protein and whether influences on diet are immediate or develop over time. To determine the effects of protein restriction on consumption, preference, and palatability for protein we assessed patterns of intake for casein (protein) and maltodextrin (carbohydrate) solutions in adult rats. To induce a state of protein restriction, rats were maintained on a low protein diet (5% casein) and compared to control rats on non-restricted diet (20% casein). Under these dietary conditions, relative to control rats, protein-restricted rats exhibited hyperphagia without weight gain. After two weeks, on alternate conditioning days, rats were given access to either isocaloric casein or maltodextrin solutions that were saccharin-sweetened and distinctly flavoured whilst consumption and licking patterns were recorded. This allowed rats to learn about the post-ingestive nutritional consequences of the two different solutions. Subsequently, during a preference test when rats had access to both solutions, we found that protein-restricted rats exhibited a preference for casein over carbohydrate whereas non-restricted rats did not. Analysis of lick microstructure revealed that this preference was associated with an increase in cluster size and number, reflective of an increase in palatability. In conclusion, protein-restriction induced a conditioned preference for protein, relative to carbohydrate, and this was associated with increased palatability.

neuroscience↗