Callahan, K. P. | Search bioRxiv

bioRxiv2022

Mouse primary T cell phosphotyrosine proteomics enabled by BOOST

The Broad Spectrum Optimization of Selective Triggering (BOOST) approach was recently developed to increase the quantitative depth of the tyrosine phosphoproteome by mass spectrometry-based proteomics. While BOOST has been demonstrated in the Jurkat T cell line, it has not been demonstrated in scarce mice primary T cells. Here, we show the first phosphotyrosine proteomics experiment performed in mice primary T cells using BOOST. We identify and precisely quantify more than 2,000 unique pTyr sites from more than 3,000 unique pTyr peptide PSMs using only 1 mg of protein from T cell receptor-stimulated primary T cells from mice. We further reveal the importance of the phase-constrained spectrum deconvolution method ({Phi}SDM) parameter on Orbitrap instruments that, when disabled, enhances quantitation depth, accuracy, and precision in low-abundance samples. Using samples with contrived ratios, we find that disabling {Phi}SDM allows for up to a two-fold increase in the number of statistically significant intensity ratios detected while enabling {Phi}SDM degrades quantitation, especially in low-abundance samples. TOC Graphic O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=111 SRC="FIGDIR/small/491817v1_ufig1.gif" ALT="Figure 1"> View larger version (27K): org.highwire.dtl.DTLVardef@4a2742org.highwire.dtl.DTLVardef@57645borg.highwire.dtl.DTLVardef@17dbfacorg.highwire.dtl.DTLVardef@b2df00_HPS_FORMAT_FIGEXP M_FIG C_FIG

biochemistry↗

bioRxiv2021

SILAC phosphoproteomics reveals unique signaling circuits in CAR-T cells and the inhibition of B cell-activating phosphorylation in target cells

Chimeric antigen receptor (CAR) is a single-pass transmembrane receptor designed to specifically target and eliminate cancers. While CARs prove highly efficacious against B cell malignancies, the intracellular signaling events which promote CAR T cell activity remain elusive. To gain further insight into both CAR T cell signaling and the potential signaling response of cells targeted by CAR, we analyzed phosphopeptides captured by two separate phopshoenrichment strategies from third generation CD19-CAR T cells cocultured with SILAC labeled Raji B cells by liquid chromatography-tandem mass spectrometry (LC-MS/MS). Here, we report that CD19-CAR T cells upregulated several key phosphorylation events also observed in canonical T cell receptor (TCR) signaling while Raji B cells exhibited a significant decrease in B cell receptor-signaling related phosphorylation events in response to coculture. Our data suggest that CD19-CAR stimulation activates a mixture of unique CD19-CAR-specific signaling pathways and canonical TCR signaling while global phosphorylation in Raji B cells is reduced after association with the CD19-CAR T cells.

cell biology↗

bioRxiv2019

Spontaneous mutations that confer resistance to 2-deoxyglucose act through Hxk2 and Snf1 pathways to regulate gene expression and HXT endocytosis

Yeast and fast-growing human tumor cells share metabolic similarities in that both cells use fermentation of glucose for energy and both are highly sensitive to the glucose analog 2-deoxyglucose. Spontaneous mutations in S. cerevisiae that conferred resistance to 2-deoxyglucose were identified by whole genome sequencing. In addition to three aneuploid strains, we detected missense alleles of the HXK2, REG1, GLC7 and SNF1 genes that were shown to confer significant resistance to 2-deoxyglucose. All three missense alleles in HXK2 resulted in significantly reduced catalytic activity. Missense alleles affecting the Snf1 kinase pathway (REG1, GLC7 and SNF1) exhibited different capacities to affect the regulation of invertase expression. Of the seven missense alleles identified in this study, all but one affected hexose transporter endocytosis by increasing plasma membrane occupancy of the Hxt3 protein. Increased expression of the DOG (deoxyglucose) phosphatases has been associated with resistance to 2-deoxyglucose. Expression of both the DOG1 and DOG2 mRNA was elevated after treatment with 2-deoxyglucose. Deletion of the HXK2 and REG1 genes confers resistance to 2-deoxyglucose and causes increased expression of the DOG2 mRNA. We conclude that Snf1 kinase-mediated regulation of the endocytosis of the hexose transporters and regulation of DOG2 expression are important mechanisms for resistance to 2-deoxyglucose. However, the dominant SNF1-G53R allele can confer additional 2-deoxyglucose resistance in cells that are genetically compromised in both the endocytosis and DOG pathways. Thus at least one more mechanism for conferring resistance to this glucose analog remains to be discovered.\n\nAuthor SummaryYeast and fast-growing human tumor cells share metabolic similarities in that both cells use fermentation of glucose for energy and both are highly sensitive to the glucose analog 2-deoxyglucose. Another similarity between yeast cells and human tumor cells is that both cells can acquire resistance to 2-deoxyglucose, an outcome that can limit the usefulness of some cancer therapeutics. In this study, we used bakers yeast as a model organism to better understand the mechanism of toxicity and acquisition of resistance to 2-deoxyglucose. Spontaneous mutations in S. cerevisiae that conferred resistance to 2-deoxyglucose were isolated and identified by whole genome sequencing, a technology that was not available until recently. Our studies indicate that 2-deoxyglucose becomes toxic after it is phosphorylated by an enzyme called hexokinase. One important route to resistance is to reduce hexokinase activity. Other parallel pathways to resistance include increased expression of a hydrolase that degrades the toxic metabolite, altered localization of glucose transporters and altered glucose signal transduction pathways.

genetics↗