Deep phosphotyrosine characterisation of primary murine T cells using broad spectrum optimisation of selective triggering.

Sequencing the tyrosine phosphoproteome using MS-based proteomics is challenging due to the low abundance of tyrosine phosphorylation in cells, a challenge compounded in scarce samples like primary cells or clinical samples. The broad-spectrum optimisation of selective triggering (BOOST) method was recently developed to increase phosphotyrosine sequencing in low protein input samples by leveraging tandem mass tags (TMT), phosphotyrosine enrichment, and a phosphotyrosine-loaded carrier channel. Here, we demonstrate the viability of BOOST in T cell receptor (TCR)-stimulated primary murine T cells by benchmarking the accuracy and precision of the BOOST method and discerning significant alterations in the phosphoproteome associated with receptor stimulation.

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 phosphoenrichment strategies from third generation CD19-CAR T cells cocultured with SILAC labeled Raji B cells by liquid chromatography-tandem mass spectrometry (LC-MS/MS).

Ovalbumin Antigen-Specific Activation of Human T Cell Receptor Closely Resembles Soluble Antibody Stimulation as Revealed by BOOST Phosphotyrosine Proteomics.

Activation of the T cell receptor (TCR) leads to a network of early signaling predominantly orchestrated by tyrosine phosphorylation in T cells. The TCR is commonly activated using soluble anti-TCR antibodies, but this approach is not antigen-specific. Alternatively, activating the TCR using specific antigens of a range of binding affinities in the form of a peptide-major histocompatibility complex (pMHC) is presumed to be more physiological.

Quantitative Interactomics of Lck-TurboID in Living Human T Cells Unveils T Cell Receptor Stimulation-Induced Proximal Lck Interactors.

While Lck has been widely recognized to play a pivotal role in the initiation of the T cell receptor (TCR) signaling pathway, an understanding of the precise regulation of Lck in T cells upon TCR activation remains elusive. Investigation of protein-protein interaction (PPI) using proximity labeling techniques such as TurboID has the potential to provide valuable molecular insights into Lck regulatory networks. By expressing Lck-TurboID in Jurkat T cells, we have uncovered a dynamic, short-range Lck protein interaction network upon 30 min of TCR stimulation.

Tandem Mass Tag Approach Utilizing Pervanadate BOOST Channels Delivers Deeper Quantitative Characterization of the Tyrosine Phosphoproteome.

Dynamic tyrosine phosphorylation is fundamental to a myriad of cellular processes. However, the inherently low abundance of tyrosine phosphorylation in the proteome and the inefficient enrichment of phosphotyrosine(pTyr)-containing peptides has led to poor pTyr peptide identification and quantitation, critically hindering researchers' ability to elucidate signaling pathways regulated by tyrosine phosphorylation in systems where cellular material is limited. The most popular approaches to wide-scale characterization of the tyrosine phosphoproteome use pTyr enrichment with pan-specific, anti-pTyr antibodies from a large amount of starting material.

Regulation of Respiration and Apoptosis by Cytochrome c Threonine 58 Phosphorylation.

Cytochrome c (Cytc) is a multifunctional protein, acting as an electron carrier in the electron transport chain (ETC), where it shuttles electrons from bc complex to cytochrome c oxidase (COX), and as a trigger of type II apoptosis when released from the mitochondria. We previously showed that Cytc is regulated in a highly tissue-specific manner: Cytc isolated from heart, liver, and kidney is phosphorylated on Y97, Y48, and T28, respectively. Here, we have analyzed the effect of a new Cytc phosphorylation site, threonine 58, which we mapped in rat kidney Cytc by mass spectrometry.

Serine-47 phosphorylation of cytochrome in the mammalian brain regulates cytochrome oxidase and caspase-3 activity.

Cytochrome (Cyt) is a multifunctional protein that operates as an electron carrier in the mitochondrial electron transport chain and plays a key role in apoptosis. We have previously shown that tissue-specific phosphorylations of Cyt in the heart, liver, and kidney play an important role in the regulation of cellular respiration and cell death. Here, we report that Cyt purified from mammalian brain is phosphorylated on S47 and that this phosphorylation is lost during ischemia.

Fibroblasts Mobilize Tumor Cell Glycogen to Promote Proliferation and Metastasis.

Successful metastasis requires the co-evolution of stromal and cancer cells. We used stable isotope labeling of amino acids in cell culture coupled with quantitative, label-free phosphoproteomics to study the bidirectional signaling in ovarian cancer cells and human-derived, cancer-associated fibroblasts (CAFs) after co-culture. In cancer cells, the interaction with CAFs supported glycogenolysis under normoxic conditions and induced phosphorylation and activation of phosphoglucomutase 1, an enzyme involved in glycogen metabolism.

Lck promotes Zap70-dependent LAT phosphorylation by bridging Zap70 to LAT.

T cell-antigen receptor (TCR) signaling requires the sequential activities of the kinases Lck and Zap70. Upon TCR stimulation, Lck phosphorylates the TCR, thus leading to the recruitment, phosphorylation, and activation of Zap70. Lck binds and stabilizes phosho-Zap70 by using its SH2 domain, and Zap70 phosphorylates the critical adaptors LAT and SLP76, which coordinate downstream signaling.

Sucrose Nonfermenting-Related Kinase Regulates Both Adipose Inflammation and Energy Homeostasis in Mice and Humans.

Sucrose nonfermenting-related kinase (SNRK) is a member of the AMPK-related kinase family, and its physiological role in adipose energy homeostasis and inflammation remains unknown. We previously reported that SNRK is ubiquitously and abundantly expressed in both white adipose tissue (WAT) and brown adipose tissue (BAT), but SNRK expression diminishes in adipose tissue in obesity. In this study we report novel experimental findings from both animal models and human genetics.

A PLC-γ1 Feedback Pathway Regulates Lck Substrate Phosphorylation at the T-Cell Receptor and SLP-76 Complex.

Phospholipase C gamma 1 (PLC-γ1) occupies a critically important position in the T-cell signaling pathway. While its functions as a regulator of both Ca signaling and PKC-family kinases are well characterized, PLC-γ1's role in the regulation of early T-cell receptor signaling events is incompletely understood. Activation of the T-cell receptor leads to the formation of a signalosome complex between SLP-76, LAT, PLC-γ1, Itk, and Vav1.

Highly reproducible improved label-free quantitative analysis of cellular phosphoproteome by optimization of LC-MS/MS gradient and analytical column construction.

Expanding the sequencing depth of the peptides with a statistically significant quantitative change derived from a biological stimulation is critical. Here we demonstrate that optimization of LC gradient and analytical column construction can reveal over 30,000 unique peptides and 23,000 phosphopeptides at high confidence. The quantitative reproducibility of different analytical workflows was evaluated by comparing the phosphoproteome of CD3/4 stimulated and unstimulated T-cells as a model system.

Quantitative Phosphoproteomic Analysis of T-Cell Receptor Signaling.

TCR signaling critically depends on protein phosphorylation across many proteins. Localization of each phosphorylation event relative to the T-cell receptor (TCR) and canonical T-cell signaling proteins will provide clues about the structure of TCR signaling networks. Quantitative phosphoproteomic analysis by mass spectrometry provides a wide-scale view of cellular phosphorylation networks.

Proteomic analysis of laser capture microdissected focal lesions in a rat model of progenitor marker-positive hepatocellular carcinoma.

We have shown previously that rapamycin, the canonical inhibitor of the mechanistic target of rapamycin (mTOR) complex 1, markedly inhibits the growth of focal lesions in the resistant hepatocyte (Solt-Farber) model of hepatocellular carcinoma (HCC) in the rat. In the present study, we characterized the proteome of persistent, pre-neoplastic focal lesions in this model. One group was administered rapamycin by subcutaneous pellet for 3 weeks following partial hepatectomy and euthanized 4 weeks after the cessation of rapamycin.

Phosphorylation of Cytochrome c Threonine 28 Regulates Electron Transport Chain Activity in Kidney: IMPLICATIONS FOR AMP KINASE.

Mammalian cytochrome c (Cytc) plays a key role in cellular life and death decisions, functioning as an electron carrier in the electron transport chain and as a trigger of apoptosis when released from the mitochondria. However, its regulation is not well understood. We show that the major fraction of Cytc isolated from kidneys is phosphorylated on Thr, leading to a partial inhibition of respiration in the reaction with cytochrome c oxidase.

Targeted proteomics: Current status and future perspectives for quantification of food allergens.

Unlabelled: Allergen levels in fresh and processed foods can vary dynamically. As different sources of foods can cause different types of allergic reactions, the food industry and regulatory bodies urgently require reliable detection and absolute quantitation methods for allergen detection in complex food products to effectively safeguard the food-allergic population. Recent advances of targeted proteomic technologies namely multiple-reaction monitoring (MRM) mass spectrometry (MS) coupled with isotope-labeled internal standard, also known as AQUA peptides offers absolute quantitation of food allergens even at 10ppb level in a multiplex fashion.

Vav1 Regulates T-Cell Activation through a Feedback Mechanism and Crosstalk between the T-Cell Receptor and CD28.

Vav1, a Rac/Rho guanine nucleotide exchange factor and a critical component of the T-cell receptor (TCR) signaling cascade is tyrosine phosphorylated rapidly in response to T-cell activation. Vav1 has established roles in proliferation, cytokine secretion, Ca(2+) responses, and actin cytoskeleton regulation; however, its function in the regulation of phosphorylation of TCR components, including the ζ chain, the CD3 δ, ε, γ chains, and the associated kinases Lck and ZAP-70, is not well established. To obtain a more comprehensive picture of the role of Vav1 in receptor proximal signaling, we performed a wide-scale characterization of Vav1-dependent tyrosine phosphorylation events using quantitative phosphoproteomic analysis of Vav1-deficient T cells across a time course of TCR stimulation.

Adaptation of HepG2 cells to a steady-state reduction in the content of protein phosphatase 6 (PP6) catalytic subunit.

Protein phosphatase 6 (PP6) is a ubiquitous Ser/Thr phosphatase involved in an array of cellular processes. To assess the potential of PP6 as a therapeutic target in liver disorders, we attenuated expression of the PP6 catalytic subunit in HepG2 cells using lentiviral-transduced shRNA. Two PP6 knock-down (PP6KD) cell lines (90% reduction of PP6-C protein content) were studied in depth.

The catalytic activity of the kinase ZAP-70 mediates basal signaling and negative feedback of the T cell receptor pathway.

T cell activation by antigens binding to the T cell receptor (TCR) must be properly regulated to ensure normal T cell development and effective immune responses to pathogens and transformed cells while avoiding autoimmunity. The Src family kinase Lck and the Syk family kinase ZAP-70 (ζ chain-associated protein kinase of 70 kD) are sequentially activated in response to TCR engagement and serve as critical components of the TCR signaling machinery that leads to T cell activation. We performed a mass spectrometry-based phosphoproteomic study comparing the quantitative differences in the temporal dynamics of phosphorylation in stimulated and unstimulated T cells with or without inhibition of ZAP-70 catalytic activity.

Wide-scale quantitative phosphoproteomic analysis reveals that cold treatment of T cells closely mimics soluble antibody stimulation.

The activation of T lymphocytes through antigen-mediated T cell receptor (TCR) clustering is vital in regulating the adaptive immune response. Although T cell receptor signaling has been extensively studied, the fundamental mechanisms for signal initiation are not fully understood. Reduced temperatures have initiated some of the hallmarks of TCR signaling, such as increased phosphorylation and activation on ERK and calcium release from the endoplasmic reticulum, as well as coalesced the T cell membrane microdomains.

Protein networks and activation of lymphocytes.

The signal transduction pathways initiated by lymphocyte activation play a critical role in regulating host immunity. High-resolution mass spectrometry has accelerated the investigation of these complex and dynamic pathways by enabling the qualitative and quantitative investigation of thousands of proteins and phosphoproteins simultaneously. In addition, the unbiased and wide-scale identification of protein-protein interaction networks and protein kinase substrates in lymphocyte signaling pathways can be achieved by mass spectrometry-based approaches.

SRC homology 2 domain-containing leukocyte phosphoprotein of 76 kDa (SLP-76) N-terminal tyrosine residues regulate a dynamic signaling equilibrium involving feedback of proximal T-cell receptor (TCR) signaling.

SRC homology 2 domain-containing leukocyte phosphoprotein of 76 kDa (SLP-76) is a cytosolic adaptor protein that plays an important role in the T-cell receptor-mediated T-cell signaling pathway. SLP-76 links proximal receptor stimulation to downstream effectors through interaction with many signaling proteins. Previous studies showed that mutation of three tyrosine residues, Tyr(112), Tyr(128), and Tyr(145), in the N terminus of SLP-76 results in severely impaired phosphorylation and activation of Itk and PLCγ1, which leads to defective calcium mobilization, Erk activation, and NFAT activation.

Novel Cul3 binding proteins function to remodel E3 ligase complexes.

Background: Cullins belong to a family of scaffold proteins that assemble multi-subunit ubiquitin ligase complexes to recruit protein substrates for ubiquitination via unique sets of substrate adaptor, such as Skp1 or Elongin B, and a substrate-binding protein with a conserved protein-protein interacting domain, such as leucine-rich repeats (LRR), a WD40 domain, or a zinc-finger domain. In the case of the Cullin3 (Cul3), it forms a BTB-Cul3-Rbx1 (BCR) ubiquitin ligase complex where it is believed that a BTB domain-containing protein performs dual functions where it serves as both the substrate adaptor and the substrate recognition protein.

Results: Tandem affinity purification and LC/MS-MS analysis of the BCR complex led to the identification of 10,225 peptides.

Mitogen-activated protein kinase phosphatase 3 (MKP-3)-deficient mice are resistant to diet-induced obesity.

Mitogen-activated protein kinase phosphatase 3 (MKP-3) is a negative regulator of extracellular signal-related kinase signaling. Our laboratory recently demonstrated that MKP-3 plays an important role in obesity-related hyperglycemia by promoting hepatic glucose output. This study shows that MKP-3 deficiency attenuates body weight gain induced by a high-fat diet (HFD) and protects mice from developing obesity-related hepatosteatosis.