Bettina Hahn

Dynamic Mathematical Modeling of IL13-Induced Signaling in Hodgkin and Primary Mediastinal B-Cell Lymphoma Allows Prediction of Therapeutic Targets

Primary mediastinal B-cell lymphoma (PMBL) and classical Hodgkin lymphoma (cHL) share a frequent constitutive activation of JAK (Janus kinase)/STAT signaling pathway. Because of complex, nonlinear relations within the pathway, key dynamic properties remained to be identified to predict possible strategies for intervention. We report the development of dynamic pathway models based on quantitative data collected on signaling components of JAK/STAT pathway in two lymphoma-derived cell lines, MedB-1 and L1236, representative of PMBL and cHL, respectively. We show that the amounts of STAT5 and STAT6 are higher whereas those of SHP1 are lower in the two lymphoma cell lines than in normal B cells. Distinctively, L1236 cells harbor more JAK2 and less SHP1 molecules per cell than MedB-1 or control cells. In both lymphoma cell lines, we observe interleukin-13 (IL13)-induced activation of IL4 receptor α, JAK2, and STAT5, but not of STAT6. Genome-wide, 11 early and 16 sustained genes are upregulated by IL13 in both lymphoma cell lines. Specifically, the known STAT-inducible negative regulators CISH and SOCS3 are upregulated within 2 hours in MedB-1 but not in L1236 cells. On the basis of this detailed quantitative information, we established two mathematical models, MedB-1 and L1236 model, able to describe the respective experimental data. Most of the model parameters are identifiable and therefore the models are predictive. Sensitivity analysis of the model identifies six possible therapeutic targets able to reduce gene expression levels in L1236 cells and three in MedB-1. We experimentally confirm reduction in target gene expression in response to inhibition of STAT5 phosphorylation, thereby validating one of the predicted targets.

Quantitative protein microarrays for time-resolved measurements of protein phosphorylation

The quantitative analysis of signaling networks requires highly sensitive methods for the time‐resolved determination of protein phosphorylation. For this reason, we developed a quantitative protein microarray that monitors the activation of multiple signaling pathways in parallel, and at high temporal resolution. A label‐free sandwich approach was combined with near infrared detection, thus permitting the accurate quantification of low‐level phosphoproteins in limited biological samples corresponding to less than 50 000 cells, and with a very low standard deviation of approximately 5%. The identification of suitable antibody pairs was facilitated by determining their accuracy and dynamic range using our customized software package Quantpro. Thus, we are providing an important tool to generate quantitative data for systems biology approaches, and to drive innovative diagnostic applications.

Phosphorus-based absolutely quantified standard peptides for quantitative proteomics.

An innovative method for the production of absolutely quantified peptide standards is described. These are named phosphorus-based absolutely quantified standard (PASTA) peptides. As the first step, synthetic phosphopeptides are calibrated via a hybrid LC-(ICP+ESI)-MS system. Quantification is achieved by ICP-MS detection of 31P, and identification is performed by ESI-MS. Generation of phosphopeptide standard solutions with this system is demonstrated to provide absolute concentrations with an accuracy better than 10%. The concept was extended to the production of peptide standards by subjecting a PASTA phosphopeptide to gentle and complete dephosphorylation to obtain the cognate PASTA peptide. It is demonstrated that both enzymatic hydrolysis by alkaline or antarctic phosphatase or chemical hydrolysis by hydrofluoric acid can be employed for this purpose. Further, the introduction of one or more stable isotope-labeled amino acids (preferably labeled by 13C, 15N) results in the production of a labeled PASTA peptide, which then can be employed as an internal standard for quantitative analysis by LC-ESI-MS. Using a 1:1 mixture of a stable isotope-labeled PASTA peptide/phosphopeptide pair as dual standard, a quantification between active and inactive recombinant MAP kinase p38alpha was performed by a combination of tryptic digestion and nanoLC-MS.

Heterogeneous kinetics of AKT signaling in individual cells are accounted for by variable protein concentration

In most solid cancers, cells harboring oncogenic mutations represent only a sub-fraction of the entire population. Within this sub-fraction the expression level of mutated proteins can vary significantly due to cellular variability limiting the efficiency of targeted therapy. To address the causes of the heterogeneity, we performed a systematic analysis of one of the most frequently mutated pathways in cancer cells, the phosphatidylinositol 3 kinase (PI3K) signaling pathway. Among others PI3K signaling is activated by the hepatocyte growth factor (HGF) that regulates proliferation of hepatocytes during liver regeneration but also fosters tumor cell proliferation. HGF-mediated responses of PI3K signaling were monitored both at the single cell and cell population level in primary mouse hepatocytes and in the hepatoma cell line Hepa1_6. Interestingly, we observed that the HGF-mediated AKT responses at the level of individual cells is rather heterogeneous. However, the overall average behavior of the single cells strongly resembled the dynamics of AKT activation determined at the cell population level. To gain insights into the molecular cause for the observed heterogeneous behavior of individual cells, we employed dynamic mathematical modeling in a stochastic framework. Our analysis demonstrated that intrinsic noise was not sufficient to explain the observed kinetic behavior, but rather the importance of extrinsic noise has to be considered. Thus, distinct from gene expression in the examined signaling pathway fluctuations of the reaction rates has only a minor impact whereas variability in the concentration of the various signaling components even in a clonal cell population is a key determinant for the kinetic behavior.

Cellular ERK phospho-form profiles with conserved preference for a switch-like pattern.

ERK is a member of the MAPK pathway with essential functions in cell proliferation, differentiation, and survival. Complete ERK activation by the kinase MEK requires dual phosphorylation at T and Y within the activation motif TEY. We show that exposure of primary mouse hepatocytes to hepatocyte growth factor (HGF) results in phosphorylation at the activation motif, but not of other residues nearby. To determine the relative abundances of unphosphorylated ERK and the three ERK phospho-forms pT, pY, and pTpY, we employed an extended one-source peptide/phosphopeptide standard method in combination with nanoUPLC-MS. This method enabled us to determine the abundances of phospho-forms with a relative variability of ≤5% (SD). We observed a switch-like preference of ERK phospho-form abundances toward the active, doubly phosphorylated and the inactive, unphosphorylated form. Interestingly, ERK phospho-form profiles were similar upon growth factor and cytokine stimulation. A screening of several murine and human cell systems revealed that the balance between TY- and pTpY-ERK is conserved while the abundances of pT- and pY-ERK are more variable within cell types. We show that the phospho-form profiles do not change by blocking MEK activity suggesting that cellular phosphatases determine the ERK phospho-form distribution. This study provides novel quantitative insights into multisite phosphorylation.

One-source peptide/phosphopeptide standards for accurate phosphorylation degree determination

Reversible protein phosphorylation is a key mediator for intracellular signal transduction. Here we report an innovative method for accurate, site‐specific protein phosphorylation degree determination by nanoLC‐ESI‐MS/MS. A stable isotope‐labeled pair of peptide/phosphopeptide standards with volumetrically defined molar ratio is used as reference, providing an internal standard for both the analyte peptide and the phosphopeptide. For the preparation of one‐source peptide/phosphopeptide standards, an aliquot of the labeled phosphopeptide standard is quantitatively dephosphorylated, yielding an equimolar solution of the peptide standard. Subsequently, the two solutions are mixed at a 1:1 or other volumetric ratio, which equals the molar ratio. This procedure assures a defined concentration ratio of both components that is independent from their absolute concentration. We demonstrate the applicability of the one‐source peptide/phosphopeptide standard method by determining the phosphorylation degree of the signalling proteins STAT5A/B and STAT6.

Site-specific degree of phosphorylation in proteins measured by liquid chromatography-electrospray mass spectrometry

This review focuses on quantitative protein phosphorylation analysis based on coverage of both the phosphorylated and nonphosphorylated forms. In this way, site‐specific data on the degree of phosphorylation can be measured, generating the most detailed level of phosphorylation status analysis of proteins. To highlight the experimental challenges in this type of quantitative protein phosphorylation analysis, we discuss the typical workflows for mass spectrometry‐based proteomics with a focus on the quantitative analysis of peptide/phosphopeptide ratios. We review workflows for measuring site‐specific degrees of phosphorylation including the label‐free approach, differential stable isotope labeling of analytes, and methods based on the addition of stable isotope labeled peptide/phosphopeptide pairs as internal standards. The discussion also includes the determination of phosphopeptide isoform abundance data for multiply phosphorylated motifs that contain information about the connectivity of phosphorylation events. The review closes with a prospective on the use of intact stable isotope labeled proteins as internal standards and a summarizing discussion of the typical accuracies of the individual methods.

Context-specific flow through the MEK/ERK module produces cell- and ligand-specific patterns of ERK single and double phosphorylation.

ERK phosphorylated on a single residue of the activation motif may be a sign of dysregulated proliferation. ERK phosphorylation patterns In the ERK pathway, the dual-specificity kinase MEK phosphorylates a threonine and a tyrosine residue in ERK, and this dual-phosphorylated form is the fully active kinase. Iwamoto et al. used mass spectrometry, quantitative Western blotting, and mathematical modeling to explore MEK-dependent phosphorylation dynamics of ERK in skin and liver cells exposed to either a cytokine, IL-6, or a growth factor, HGF. Not surprisingly, the different stimuli produced different dynamics of ERK phosphorylation, and skin and liver cells responded differently to the same ligand. The dynamics of the changes in the abundance of the phosphorylated forms of ERK (pT-ERK, pY-ERK, and pTpY-ERK) and the relative distributions of the single- and double-phosphorylated forms of ERK were different. Mathematical modeling indicated that distinct network structures with or without regulated feedback loops produced the different dynamics of ERK phosphorylation and distributions of phosphorylated ERK. This study provides biochemical insight into how a single pathway can produce distinct responses, such as differentiation or proliferation. The same pathway, such as the mitogen-activated protein kinase (MAPK) pathway, can produce different cellular responses, depending on stimulus or cell type. We examined the phosphorylation dynamics of the MAPK kinase MEK and its targets extracellular signal–regulated kinase 1 and 2 (ERK1/2) in primary hepatocytes and the transformed keratinocyte cell line HaCaT A5 exposed to either hepatocyte growth factor or interleukin-6. By combining quantitative mass spectrometry with dynamic modeling, we elucidated network structures for the reversible threonine and tyrosine phosphorylation of ERK in both cell types. In addition to differences in the phosphorylation and dephosphorylation reactions, the HaCaT network model required two feedback mechanisms, which, as the experimental data suggested, involved the induction of the dual-specificity phosphatase DUSP6 and the scaffold paxillin. We assayed and modeled the accumulation of the double-phosphorylated and active form of ERK1/2, as well as the dynamics of the changes in the monophosphorylated forms of ERK1/2. Modeling the differences in the dynamics of the changes in the distributions of the phosphorylated forms of ERK1/2 suggested that different amounts of MEK activity triggered context-specific responses, with primary hepatocytes favoring the formation of double-phosphorylated ERK1/2 and HaCaT A5 cells that produce both the threonine-phosphorylated and the double-phosphorylated form. These differences in phosphorylation distributions explained the threshold, sensitivity, and saturation of the ERK response. We extended the findings of differential ERK phosphorylation profiles to five additional cultured cell systems and matched liver tumor and normal tissue, which revealed context-specific patterns of the various forms of phosphorylated ERK.

Structural and mechanistic information on c1 ion formation in collision-induced fragmentation of peptides

The formation of c1 ions during collision-induced fragmentation of peptides with asparagine, ornithine, or glutamine at the N-terminal position 2 has been studied. For this purpose, the corresponding fragment ion spectra of a large set of synthetic peptides were investigated. It is demonstrated that the c1 ion intensity depends on the nature of the second residue in the N-terminal dipeptide motif as well as on the peptide length. It is shown that the formation of c1 ions proceeds by two competing mechanisms. One mechanism is the secondary fragmentation of the b2 ion, the efficiency of which shows only a minor dependency on the complete peptide sequence. The other mechanism is the direct formation from the molecular ion, which is identified to be connected with sequence-specific c1 ion intensities. A model for this latter mechanism is proposed based on the analysis of the formation and secondary fragmentation of the zmax-1 ion, which is the complementary ion to the c1 ion. Additional evidence is obtained by investigation of peptides with ornithine in N-terminal position 2, which in general exhibit c1 ion intensities intermediate between the asparagine- and glutamine-containing species. The data presented support the reliable assignment of N-terminal dipeptide motifs using collision-induced dissociation.

IL-1-induced and p38MAPK-dependent activation of the mitogen-Activated protein kinase-Activated protein kinase 2 (MK2) in hepatocytes: Signal transduction with robust and concentration-independent signal amplification

The IL-1β induced activation of the p38MAPK/MAPK-activated protein kinase 2 (MK2) pathway in hepatocytes is important for control of the acute phase response and regulation of liver regeneration. Many aspects of the regulatory relevance of this pathway have been investigated in immune cells in the context of inflammation. However, very little is known about concentration-dependent activation kinetics and signal propagation in hepatocytes and the role of MK2. We established a mathematical model for IL-1β-induced activation of the p38MAPK/MK2 pathway in hepatocytes that was calibrated to quantitative data on time- and IL-1β concentration-dependent phosphorylation of p38MAPK and MK2 in primary mouse hepatocytes. This analysis showed that, in hepatocytes, signal transduction from IL-1β via p38MAPK to MK2 is characterized by strong signal amplification. Quantification of p38MAPK and MK2 revealed that, in hepatocytes, at maximum, 11.3% of p38MAPK molecules and 36.5% of MK2 molecules are activated in response to IL-1β. The mathematical model was experimentally validated by employing phosphatase inhibitors and the p38MAPK inhibitor SB203580. Model simulations predicted an IC50 of 1–1.2 μm for SB203580 in hepatocytes. In silico analyses and experimental validation demonstrated that the kinase activity of p38MAPK determines signal amplitude, whereas phosphatase activity affects both signal amplitude and duration. p38MAPK and MK2 concentrations and responsiveness toward IL-1β were quantitatively compared between hepatocytes and macrophages. In macrophages, the absolute p38MAPK and MK2 concentration was significantly higher. Finally, in line with experimental observations, the mathematical model predicted a significantly higher half-maximal effective concentration for IL-1β-induced pathway activation in macrophages compared with hepatocytes, underscoring the importance of cell type-specific differences in pathway regulation.