Biology subjects

Storey, A. J.

Publications and source records attributed to Storey, A. J..

5 recordsLinked to original sources

bioRxiv2025

DNA-PKcs governs LAT-dependent signaling in CD4+ and CD8+ T cells

Formation of the immune synapse (IS) following T cell antigen recognition includes recruitment of the Linker for Activation of T cells (LAT). Once at the IS, LAT tyrosines are phosphorylated allowing it to serve as a scaffold for formation of the "signalosome", a multiprotein complex that drives TCR signaling. Here, we show that upon T cell activation, DNA dependent protein kinase catalytic subunit (DNA-PKcs) interacts with LAT and localizes to the IS. Inhibition of DNA-PKcs diminishes LAT localization at the IS. We identified two LAT serines phosphorylated by DNA-PKcs, S224 and S241, that impact LAT tyrosine phosphorylation, protein binding, and cytokine production. Using our mouse model designed to delete DNA-PKcs expression within mature CD4+ or CD8+ T cells, we show loss of DNA-PKcs results in T cells unable to control tumor growth or induce allogeneic graft rejection. These data bring to the forefront DNA-PKcs as a pivotal protein in T cell function.

immunology↗

bioRxiv2025

Dynamic global acetylation remodeling during the yeast heat shock response

All organisms experience stress and must rapidly respond to changing conditions. Thus, cells have evolved sophisticated rapid-response mechanisms such as post-translational protein modification to rapidly and reversibly modulate protein activity. One such post-translational modification is reversible lysine acetylation, where proteomic studies have identified thousands of acetylated proteins across diverse organisms. While the sheer size of the acetylome is striking, the function of acetylation for the vast majority of proteins remains largely obscure. Here, we show that global acetylation plays a previously unappreciated role in the heat shock response of Saccharomyces cerevisiae. We find that dysregulated acetylation renders cells heat sensitive, and moreover, that the acetylome is globally remodeled during heat shock over time. Using quantitative acetyl-proteomics, we identified [~]400 high-confidence acetyl marks across [~]200 proteins that significantly change in acetylation when cells are shifted to elevated temperature. Proteins with significant changes in lysine acetylation during heat shock strongly overlap with genes induced or repressed by stress. Thus, we hypothesize that protein acetylation augments the heat shock response by activating induced proteins and inactivating repressed proteins. Intriguingly, we find nearly 40 proteins with at least two acetyl marks that significantly change in the opposite directions. These proteins are strongly enriched for chaperones and ribosomal proteins, suggesting that these two key processes are coordinately regulated by protein acetylation during heat shock. Moreover, we hypothesize that the same type of activating and inactivating marks that exist on histones may be a general feature of proteins regulated by acetylation. Overall, this work has identified a new layer of post-translational regulation that likely augments the classic heat shock response.

genomics↗

bioRxiv2023

Characterization of methionine dependence in melanoma cells

Dietary methionine restriction is associated with a reduction in tumor growth in preclinical studies and an increase in lifespan in animal models. The mechanism by which methionine restriction inhibits tumor growth while sparing normal cells is incompletely understood. We do know that normal cells can utilize methionine or homocysteine interchangeably (methionine independence) while most cancer cells are strictly dependent on methionine availability. Here, we compared a typical methionine dependent and a rare methionine independent melanoma cell line. We show that replacing methionine, a methyl donor, with its precursor homocysteine generally induced hypomethylation in gene promoters. This decrease was similar in methionine dependent and methionine independent cells. There was only a low level of pathway enrichment, suggesting that the hypomethylation is generalized rather than gene specific. Whole proteome and transcriptome were also analyzed. This analysis revealed that contrarily to the effect on methylation, the replacement of methionine with homocysteine had a much greater effect on the transcriptome and proteome of methionine dependent cells than methionine independent cells. Interestingly, methionine adenosyltransferase 2A (MAT2A), responsible for the synthesis of s-adenosylmethionine from methionine, was equally strongly upregulated in both cell lines. This suggests that the absence of methionine is equally detected but triggers different outcomes in methionine dependent versus independent cells. Our analysis reveals the importance of cell cycle control, DNA damage repair, translation, nutrient sensing, oxidative stress and immune functions in the cellular response to methionine stress in melanoma.

molecular biology↗

bioRxiv2021

DNA-PKcs kinase activity stabilizes the transcription factor Egr1 in activated immune cells

DNA-dependent protein kinase catalytic subunit (DNA-PKcs) is known primarily for its function in DNA double-stranded break repair and non-homologous end joining (NHEJ). However, DNA-PKcs also has a critical yet undefined role in immunity impacting both myeloid and lymphoid cell lineages spurring interest in targeting DNA-PKcs for therapeutic strategies in immune-related diseases. To gain insight into the function of DNA-PKcs within immune cells, we performed a quantitative phosphoproteomic screen in T cells to identify first order phosphorylation targets of DNA-PKcs. Results indicate that DNA-PKcs phosphorylates the transcription factor Egr1 (early growth response protein 1) at S301. Expression of Egr1 is induced early upon T cell activation and dictates T cell response by modulating expression of cytokines and key costimulatory molecules. Mutation of serine 301 to alanine via CRISPR-Cas9 resulted in increased proteasomal degradation of Egr1 and a decrease in Egr1-dependent transcription of IL2 (interleukin-2) in activated T cells. Our findings identify DNA-PKcs as a critical intermediary link between T cell activation and T cell fate and a novel phosphosite involved in regulating Egr1 activity.

molecular biology↗

bioRxiv2019

Accurate and Sensitive Quantitation of the Dynamic Heat Shock Proteome using Tandem Mass Tags

Cells respond to environmental perturbations and insults through modulating protein abundance and function. However, the majority of studies have focused on changes in RNA abundance because quantitative transcriptomics has historically been more facile than quantitative proteomics. Modern Orbitrap mass spectrometers now provide sensitive and deep proteome coverage, allowing direct, global quantification of not only protein abundance, but also post-translational modifications (PTMs) that regulate protein activity. We implemented, and validated using the well-characterized heat shock response of budding yeast, a tandem mass tagging (TMT), triple-stage mass spectrometry (MS3) strategy to measure global changes in the proteome during the yeast heat shock response over nine timepoints. We report that basic pH, ultra-high performance liquid chromatography (UPLC) fractionation of tryptic peptides yields superfractions of minimal redundancy, a crucial requirement for deep coverage and quantification by subsequent LC-MS3. We quantified 2,275 proteins across 3 biological replicates, and found that differential expression peaked near 90 minutes following heat shock (with 868 differentially expressed proteins at 5% FDR). The sensitivity of the approach also allowed us to detect changes in the relative abundance of ubiquitination and phosphorylation PTMs over time. Remarkably, relative quantification of post-translationally modified peptides revealed striking evidence of regulation of the heat shock response by protein PTMs. These data demonstrate that the high precision of TMT-MS3 enables peptide-level quantification of samples, which can reveal important regulation of protein abundance and regulatory PTMs under various experimental conditions.

genomics↗