Inferring the Sign of Kinase-Substrate Interactions by Combining Quantitative Phosphoproteomics with a Literature-Based Mammalian Kinome Network

Protein phosphorylation is a reversible post-translational modification commonly used by cell signaling networks to transmit information about the extracellular environment into intracellular organelles for the regulation of the activity and sorting of proteins within the cell. For this study we reconstructed a literature-based mammalian kinase-substrate network from several online resources. The interactions within this directed graph network connect kinases to their substrates, through specific phosphosites including kinase-kinase regulatory interactions. However, the "signs" of links, activation or inhibition of the substrate upon phosphorylation, within this network are mostly unknown. Here we show how we can infer the "signs" indirectly using data from quantitative phosphoproteomics experiments applied to mammalian cells combined with the literature-based kinase-substrate network. Our inference method was able to predict the sign for 321 links and 153 phosphosites on 120 kinases, resulting in signed and directed subnetwork of mammalian kinase-kinase interactions. Such an approach can rapidly advance the reconstruction of cell signaling pathways and networks regulating mammalian cells.Comment: 5 page, 3 figures, IEEE-BIBE confrenc

Structure and Function Study of the Rhodobacter Sphaeroides Cytochrome Bc Complex

Biochemistry and Molecular Biolog

Diagnosis of T-cell-mediated kidney rejection by biopsy-based proteomic biomarkers and machine learning

BackgroundBiopsy-based diagnosis is essential for maintaining kidney allograft longevity by ensuring prompt treatment for graft complications. Although histologic assessment remains the gold standard, it carries significant limitations such as subjective interpretation, suboptimal reproducibility, and imprecise quantitation of disease burden. It is hoped that molecular diagnostics could enhance the efficiency, accuracy, and reproducibility of traditional histologic methods.MethodsQuantitative label-free mass spectrometry analysis was performed on a set of formalin-fixed, paraffin-embedded (FFPE) biopsies from kidney transplant patients, including five samples each with diagnosis of T-cell-mediated rejection (TCMR), polyomavirus BK nephropathy (BKPyVN), and stable (STA) kidney function control tissue. Using the differential protein expression result as a classifier, three different machine learning algorithms were tested to build a molecular diagnostic model for TCMR.ResultsThe label-free proteomics method yielded 800-1350 proteins that could be quantified with high confidence per sample by single-shot measurements. Among these candidate proteins, 329 and 467 proteins were defined as differentially expressed proteins (DEPs) for TCMR in comparison with STA and BKPyVN, respectively. Comparing the FFPE quantitative proteomics data set obtained in this study using label-free method with a data set we previously reported using isobaric labeling technology, a classifier pool comprised of features from DEPs commonly quantified in both data sets, was generated for TCMR prediction. Leave-one-out cross-validation result demonstrated that the random forest (RF)-based model achieved the best predictive power. In a follow-up blind test using an independent sample set, the RF-based model yields 80% accuracy for TCMR and 100% for STA. When applying the established RF-based model to two public transcriptome datasets, 78.1%-82.9% sensitivity and 58.7%-64.4% specificity was achieved respectively.ConclusionsThis proof-of-principle study demonstrates the clinical feasibility of proteomics profiling for FFPE biopsies using an accurate, efficient, and cost-effective platform integrated of quantitative label-free mass spectrometry analysis with a machine learning-based diagnostic model. It costs less than 10 dollars per test

BiPS, a Photocleavable, Isotopically Coded, Fluorescent Cross-linker for Structural Proteomics

Cross-linking combined with mass spectrometry is an emerging approach for studying protein structure and protein-protein interactions. However, unambiguous mass spectrometric identification of cross-linked peptides derived from proteolytically digested cross-linked proteins is still challenging. Here we describe the use of a novel cross-linker, bimane bisthiopropionic aci

Copper is required for oncogenic BRAF signalling and tumorigenesis

The BRAF kinase is mutated, typically V600E, to induce an active oncogenic state in a large fraction of melanoma, thyroid, hairy cell leukemia, and to a lesser extent, a wide spectrum of other cancers1,2. BRAFV600E phosphorylates and activates the kinases MEK1 and MEK2, which in turn phosphorylate and activate the kinases ERK1 and ERK2, stimulating the MAPK pathway to promote cancer3. Targeting MEK1/2 is proving to be an important therapeutic strategy, as a MEK1/2 inhibitor provides a survival advantage in metastatic melanoma4, which is increased when co-administered with a BRAFV600E inhibitor5. In this regard, we previously found that copper (Cu) influx enhances MEK1 phosphorylation of ERK1/2 through a Cu-MEK1 interaction6. We now show that genetic loss of the high affinity Cu transporter Ctr1 or mutations in MEK1 that disrupt Cu binding reduced BRAFV600E-driven signaling and tumorigenesis. Conversely, a MEK1-MEK5 chimera that phosphorylates ERK1/2 independent of Cu or an active ERK2 restored tumor growth to cells lacking Ctr1. Importantly, Cu chelators used in the treatment of Wilson disease7 reduced tumor growth of both BRAFV600E-transformed cells and cells resistant to BRAF inhibition. Taken together, these results suggest that Cu-chelation therapy could be repurposed to treat BRAFV600E mutation-positive cancers

Understanding of the Structural Chemistry in the Uranium Oxo-Tellurium System under HT/HP Conditions

The study of phase formation in the U-Te-O systems with mono and divalent cations under high-temperature high-pressure (HT/HP) conditions has resulted in four new inorganic compounds: K2[(UO2)(Te2O7)], Mg[(UO2)(TeO3)2], Sr[(UO2)(TeO3)2] and Sr[(UO2)(TeO5)]. Tellurium occurs as TeIV, TeV, and TeVI in these phases which demonstrate the high chemical flexibility of the system. Uranium (VI) adopts a variety of coordinations, namely, UO6 in K2[(UO2)(Te2O7), UO7 in Mg[(UO2)(TeO3)2] and Sr[(UO2)(TeO3)2], and UO8 in Sr[(UO2)(TeO5)]. The structure of K2[(UO2)(Te2O7)] is featured with one dimensional (1D) [Te2O7]4- chains along the c-axis. The Te2O7 chains are further linked by UO6 polyhedra, forming the 3D [(UO2)(Te2O7)]2- anionic frameworks. In Mg[(UO2)(TeO3)2], TeO4 disphenoids share common corners with each other resulting in infinite 1D chains of [(TeO3)2]4- propagating along the a-axis. These chains link the uranyl bipyramids by edge sharing along two edges of the disphenoids, resulting in the 2D layered structure of [(UO2)(Te2O6)]2-. The structure of Sr[(UO2)(TeO3)2] is based on 1D chains of [(UO2)(TeO3)2]∞2− propagating into the c-axis. These chains are formed by edge-sharing uranyl bipyramids which are additionally fused together by two TeO4 disphenoids, which also share two edges. The 3D framework structure of Sr[(UO2)(TeO5)] is composed of 1D [TeO5]4− chains sharing edges with UO7 bipyramids. Three tunnels based on 6-Membered rings (MRs) are propagating along [001], [010] and [100] directions. The HT/HP synthetic conditions for the preparation of single crystalline samples and their structural aspects are discussed in this work

Origins of PDZ binding specificity. A computational and experimental study using NHERF1 and the parathyroid hormone receptor

Na/H exchanger regulatory factor-1 (NHERF1) is a scaffolding protein containing two PSD95/discs large protein/ZO1 (PDZ) domains that modifies the signaling, trafficking, and function of the parathyroid hormone receptor (PTHR), a family B G-protein-coupled receptor. PTHR and NHERF1 bind through a PDZ-ligand-recognition mechanism. We show that PTH elicits phosphorylation of Thr591 in the canonical -ETVM binding motif of PTHR. Conservative substitution of Thr591 with Cys does not affect PTH(1-34)-induced cAMP production or binding of PTHR to NHERF1. The findings suggested the presence of additional sites upstream of the PDZ-ligand motif through which the two proteins interact. Structural determinants outside the canonical NHERF1 PDZ-PTHR interface that influence binding have not been characterized. We used molecular dynamics (MD) simulation to predict residues involved in these interactions. Simulation data demonstrate that the negatively charged Glu side chains at positions -3, -5, and -6 upstream of the PDZ binding motif are involved in PDZ-PTHR recognition. Engineered mutant peptides representing the PTHR C-terminal region were used to measure the binding affinity with NHERF1 PDZ domains. Comparable micromolar affinities for peptides of different length were confirmed by fluorescence polarization, isothermal titration calorimetry, and surface plasmon resonance. Binding affinities measured for Ala variants validate MD simulations. The linear relation between the change in enthalpy and entropy following Ala substitutions at upstream positions -3, -5, and -6 of the PTHR peptide provides a clear example of the thermodynamic compensation rule. Overall, our data highlight sequences in PTHR that contribute to NHERF1 interaction and can be altered to prevent phosphorylation-mediated inhibition

Convergent signaling pathways regulate parathyroid hormone and fibroblast growth factor-23 action on NPT2A-mediated phosphate transport

Parathyroid hormone (PTH) and FGF23 are the primary hormones regulating acute phosphate homeostasis. Human renal proximal tubule cells (RPTECs) were used to characterize the mechanism and signaling pathways of PTH and FGF23 on phosphate transport and the role of the PDZ protein NHERF1 in mediating PTH and FGF23 effects. RPTECs express the NPT2A phosphate transporter, αKlotho, FGFR1, FGFR3, FGFR4, and the PTH receptor. FGFR1 isoforms are formed from alternate splicing of exon 3 and of exon 8 or 9 in Ir-like loop 3. Exon 3 was absent, but mRNA containing both exons 8 and 9 is present in cytoplasm. Using an FGFR1c-specific antibody together with mass spectrometry analysis, we show that RPTECs express FGFR-β1C. The data are consistent with regulated FGFR1 splicing involving a novel cytoplasmic mechanism. PTH and FGF23 inhibited phosphate transport in a concentration-dependent manner. At maximally effective concentrations, PTH and FGF23 equivalently decreased phosphate uptake and were not additive, suggesting a shared mechanism of action. Protein kinase A or C blockade prevented PTHbut notFGF23actions. Conversely, inhibitingSGK1, blocking FGFR dimerization, or knocking down Klotho expression disruptedFGF23actions but did not interfere withPTHeffects. C-terminal FGF23(180-251) competitively and selectively blocked FGF23 action without disruptingPTHeffects. However, bothPTH and FGF23-sensitive phosphate transport were abolished by NHERF1 shRNA knockdown. Extended treatment with PTH or FGF23 down-regulated NPT2A without affecting NHERF1. We conclude that FGFR1c and PTHR signaling pathways converge on NHERF1 to inhibit PTH- and FGF23-sensitive phosphate transport and down-regulate NPT2A.Fil: Sneddon, W. Bruce. University of Pittsburgh; Estados UnidosFil: Ruiz, Giovanni W.. University of Pittsburgh; Estados UnidosFil: Gallo, Luciana Ines. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. University of Pittsburgh; Estados UnidosFil: Xiao, Kunhong. University of Pittsburgh; Estados UnidosFil: Zhang, Qiangmin. University of Pittsburgh; Estados UnidosFil: Rbaibi, Youssef. University of Pittsburgh; Estados UnidosFil: Weisz, Ora A.. University of Pittsburgh; Estados UnidosFil: Apodaca, Gerard L.. University of Pittsburgh; Estados UnidosFil: Friedman, Peter A.. University of Pittsburgh; Estados Unido