Motif-centric phosphoproteomics to target kinase-mediated signaling pathways

細胞内リン酸化修飾の大規模計測に成功 --極微量試料からのリン酸化経路解析も可能に--. 京都大学プレスリリース. 2022-02-01.Identifying cellular phosphorylation pathways based on kinase-substrate relationships is a critical step to understanding the regulation of physiological functions in cells. Mass spectrometry-based phosphoproteomics workflows have made it possible to comprehensively collect information on individual phosphorylation sites in a variety of samples. However, there is still no generic approach to uncover phosphorylation networks based on kinase-substrate relationships in rare cell populations. Here, we describe a motif-centric phosphoproteomics approach combined with multiplexed isobaric labeling, in which in vitro kinase reactions are used to generate targeted phosphopeptides, which are spiked into one of the isobaric channels to increase detectability. Proof-of-concept experiments demonstrate selective and comprehensive quantification of targeted phosphopeptides by using multiple kinases for motif-centric channels. More than 7, 000 tyrosine phosphorylation sites were quantified from several tens of micrograms of starting materials. This approach enables the quantification of multiple phosphorylation pathways under physiological or pathological regulation in a motif-centric manner

Deep Learning for Spin-Orbit Torque Characterizations with a Projected Vector Field Magnet

Spin-orbit torque characterizations on magnetic heterostructures with perpendicular anisotropy are demonstrated on a projected vector field magnet via hysteresis loop shift measurement and harmonic Hall measurement with planar Hall correction. Accurate magnetic field calibration of the vector magnet is realized with the help of deep learning models, which are able to capture the nonlinear behavior between the generated magnetic field and the currents applied to the magnet. The trained models can successfully predict the applied current combinations under the circumstances of magnetic field scans, angle scans, and hysteresis loop shift measurements. The validity of the models is further verified, complemented by the comparison of the spin-orbit torque characterization results obtained from the deep-learning-trained vector magnet system with those obtained from a conventional setup comprised of two separated electromagnets. The damping-like spin-orbit torque (DL-SOT) efficiencies (|$\xi_{DL}$|) extracted from the vector magnet and the traditional measurement configuration are consistent, where |$\xi_{DL}$| $\approx$ 0.22 for amorphous W and |$\xi_{DL}$| $\approx$ 0.02 for $\alpha$-W. Our work provides an advanced method to meticulously control a vector magnet and to conveniently perform various spin-orbit torque characterizations

Untargeted, spectral libraryâ free analysis of dataâ independent acquisition proteomics data generated using Orbitrap mass spectrometers

Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/134139/1/pmic12370_am.pdfhttp://deepblue.lib.umich.edu/bitstream/2027.42/134139/2/pmic12370.pdfhttp://deepblue.lib.umich.edu/bitstream/2027.42/134139/3/pmic12370-sup-0001-SupplementaryInfo.pd

The potential impact of primary headache disorders on stroke risk

Distribution of PHDs. (DOC 55 kb

Drugging Chemokine Receptors: Biased CXCR3 Agonists Differentially Regulate Chemotaxis And Inflammation

Drugging Chemokine Receptors: Biased CXCR3 Agonists Differentially Regulate Chemotaxis And Inflammation Jeffrey Smith, MD, PhD Providence Portland Medical Center – Portland, OR Additional Authors: Dylan Eiger, BS; Chia-Feng Tsai, PhD; Lowell Nicholson,MD; Rachel Glenn, BS; Priya Alagesan, BS; Amanda MacLeod, MD; John Jacobs, PhD; Tujin Shi, PhD; Sudarshan Rajagopal, MD,PhD Introduction: G protein-coupled receptors (GPCRs) are the largest class of transmembrane receptors and the target of ~30% of FDA approved drugs. It is now well established that GPCRs can signal through multiple transducers, including classical heterotrimeric G proteins but also GPCR kinases and β-arrestins (1). While these signaling pathways can be activated or blocked by ‘balanced’ agonists or antagonists, they can also be selectively activated in ‘biased’ responses. This new GPCR signaling paradigm of ‘biased signaling’ heralds drugs with increasing efficacy and fewer side effects (2). With over 50 ligands and 20 receptors, biased agonism is prominent within the chemokine system. Here, ligands and receptors bind one another with significant redundancy. For example, the GPCR CXCR3 is expressed on activated T cells and has three established endogenous ligands: CXCL9, CXCL10, and CXCL11 (3,4). Despite its role in inflammation, infectious disease, and cancer (5), no approved drugs target CXCR3. The purpose of my research is to measure biased signaling at CXCR3 and assess the therapeutic potential of selectively targeting certain CXCR3 signaling pathways with biased agonists. Methods: Utilizing state-of-the-art transcriptomic and phosphoproteomic analyses (6), we show vast differences in CXCR3-regulated intracellular signaling. Responses were compared between vehicle, CXCL9, CXCL10, or CXCL11 treatment assessing \u3e5,000 unique phosphopeptides and \u3e13,000 genes. Biased responses were assessed in both immortalized cell lines and primary human T cells. Utilizing various second messenger reporter systems and bioluminescence resonance energy transfer assays (2,3), we identify an important proximal GPCR signaling pathway, β-arrestin, and demonstrate that CXCL11 acts as a β-arrestin-biased agonist at CXCR3. Furthermore, we screened small molecules to identify a G protein-biased and a β-arrestin-biased small molecule agonist of CXCR3. We then utilized these small molecules to measure physiological readouts of inflammation and chemotaxis in both mice and patients. Results: Endogenous chemokines of CXCR3 activate divergent intracellular signaling pathways. Using both chemokines and small molecules, we show that β-arrestin pathway signaling through CXCR3 is necessary for full efficacy chemotaxis of activated T cells in both mice and patients (p\u3c0.05). In addition, a β-arrestin-biased small molecule potentiated the cutaneous inflammatory responses in wild-type mice (p\u3c0.05), but not in either β-arrestin KO (p=0.77) or CXCR3 KO (p=0.72) mice, indicating both CXCR3 and β-arrestin dependence in T cell mediated inflammatory responses. Conclusions: Here we show that CXCL9, CXCL10, and CXCL11 activate distinct CXCR3 intracellular signaling pathways with divergent physiological effects. We clearly demonstrate that the multiple CXCR3 chemokines, CXCL9, CXCL10, and CXCL11, are not redundant in their CXCR3 signaling properties. We show that non-canonical β-arrestin signaling is necessary for certain CXCR3-regulated inflammatory responses and for chemotaxis in both mice and patients. These data strongly suggest that CXCR3 biased agonists have therapeutic promise to treat inflammatory conditions. References Smith, J.S. and Rajagopal, S., 2016. The β-arrestins: multifunctional regulators of G protein-coupled receptors. Journal of Biological Chemistry, 291(17), pp.8969-8977. Smith, J.S., Lefkowitz, R.J. and Rajagopal, S., 2018. Biased signaling: from simple switches to allosteric microprocessors. Nature Reviews Drug Discovery, 17(4), p.243. Smith, J.S., Alagesan, P., Desai, N.K., Pack, T.F., Wu, J.H., Inoue, A., Freedman, N.J. and Rajagopal, S., 2017. CXC motif chemokine receptor 3 splice variants differentially activate beta-arrestins to regulate downstream signaling pathways. Molecular pharmacology, 92(2), pp.136-150. Smith, J.S., Nicholson, L.T., Suwanpradid, J., Glenn, R.A., Knape, N.M., Alagesan, P., Gundry, J.N., Wehrman, T.S., Atwater, A.R., Gunn, M.D. MacLeod, A.S., and Rajagopal, S., 2018. Biased agonists of the chemokine receptor CXCR3 differentially control chemotaxis and inflammation. Sci. Signal., 11(555), p.eaaq1075. Chow, M.T., Ozga, A.J., Servis, R.L., Frederick, D.T., Lo, J.A., Fisher, D.E., Freeman, G.J., Boland, G.M. and Luster, A.D., 2019. Intratumoral activity of the CXCR3 chemokine system is required for the efficacy of anti-PD-1 therapy. Immunity. Tsai, C.F.*, Smith, J.S.*, Krajewski, K., Zhao, R., Moghieb, A.M., Nicora, C.D., Xiong, X., Moore, R.J., Liu, T., Smith, R.D. and Jacobs, J.M., 2019. TMT labeling facilitates RPLC-MS analysis of hydrophilic phosphopeptides. Analytical chemistry.https://digitalcommons.psjhealth.org/ppmc_internal/1003/thumbnail.jp

Body Mass Index–Mortality Relationship in Severe Hypoglycemic Patients With Type 2 Diabetes

AbstractBackgroundHypoglycemia is associated with a higher risk of death. This study analyzed various body mass index (BMI) categories and mortalities of severe hypoglycemic patients with type 2 diabetes mellitus (DM) in a hospital emergency department.MethodsThe study included 566 adults with type 2 diabetes who were admitted to 1 medical center in Taiwan between 2008 and 2009 with a diagnosis of severe hypoglycemia. Mortality data, demographics, clinical characteristics and the Charlson’s Comorbidity Index were obtained from the electronic medical records. Patients were stratified into 4 study groups as determined by the National institute of Health (NiH) and World Health organization classification for BMi, and the demographics were compared using the analysis of variance and χ2 test. Kaplan-Meier’s analysis and the Cox proportional-hazards regression model were used for mortality, and adjusted hazard ratios were adjusted for each BMi category among participants.ResultsAfter controlling for other possible confounding variables, BMI <18.5 kg/m2 was independently associated with low survival rates in the Cox regression analysis of the entire cohort of type 2 DM patients who encountered a hypoglycemic event. Compared to patients with normal BMI, the mortality risk was higher (adjusted hazard ratios = 4.9; 95% confidence interval [CI] = 2.4-9.9) in underweight patients. Infection-related causes of death were observed in 101 cases (69.2%) and were the leading cause of death.ConclusionsAn independent association was observed between BMI less than 18.5 kg/m2 and mortality among type 2 DM patient with severe hypoglycemic episode. Deaths were predominantly infection related

Increasing CD44+/CD24- tumor stem cells, and upregulation of COX-2 and HDAC6, as major functions of HER2 in breast tumorigenesis

<p>Abstract</p> <p>Background</p> <p>Cancer cells are believed to arise primarily from stem cells. CD44⁺/CD24^-have been identified as markers for human breast cancer stem cells. Although, HER2 is a well known breast cancer oncogene, the mechanisms of action of this gene are not completely understood. Previously, we have derived immortal (M13SV1), weakly tumorigenic (M13SV1R2) and highly tumorigenic (M13SV1R2N1) cell lines from a breast epithelial cell type with stem cell phenotypes after successive SV40 large T-antigen transfection, X-ray irradiation and ectopic expression of HER2/C-erbB2/neu. Recently, we found that M13SV1R2 cells became non-tumorigenic after growing in a growth factor/hormone-deprived medium (R2d cells).</p> <p>Results</p> <p>In this study, we developed M13SV1R2N1 under the same growth factor/hormone-deprived condition (R2N1d cells). This provides an opportunity to analyze HER2 effect on gene expression associated with tumorigenesis by comparative study of R2d and R2N1d cells with homogeneous genetic background except HER2 expression. The results reveal distinct characters of R2N1d cells that can be ascribed to HER2: 1) development of fast-growing tumors; 2) high frequency of CD44⁺/CD24^-cells (~50% for R2N1d vs. ~10% for R2d); 3) enhanced expression of COX-2, HDAC6 mediated, respectively, by MAPK and PI3K/Akt pathways, and many genes associated with inflammation, metastasis, and angiogenesis. Furthermore, HER2 expression can be down regulated in non-adhering R2N1d cells. These cells showed longer latent period and lower rate of tumor development compared with adhering cells.</p> <p>Conclusions</p> <p>HER2 may induce breast cancer by increasing the frequency of tumor stem cells and upregulating the expression of COX-2 and HDAC6 that play pivotal roles in tumor progression.</p