Luca Simeoni

A methodology for the structural and functional analysis of signaling and regulatory networks

BackgroundStructural analysis of cellular interaction networks contributes to a deeper understanding of network-wide interdependencies, causal relationships, and basic functional capabilities. While the structural analysis of metabolic networks is a well-established field, similar methodologies have been scarcely developed and applied to signaling and regulatory networks.ResultsWe propose formalisms and methods, relying on adapted and partially newly introduced approaches, which facilitate a structural analysis of signaling and regulatory networks with focus on functional aspects. We use two different formalisms to represent and analyze interaction networks: interaction graphs and (logical) interaction hypergraphs. We show that, in interaction graphs, the determination of feedback cycles and of all the signaling paths between any pair of species is equivalent to the computation of elementary modes known from metabolic networks. Knowledge on the set of signaling paths and feedback loops facilitates the computation of intervention strategies and the classification of compounds into activators, inhibitors, ambivalent factors, and non-affecting factors with respect to a certain species. In some cases, qualitative effects induced by perturbations can be unambiguously predicted from the network scheme. Interaction graphs however, are not able to capture AND relationships which do frequently occur in interaction networks. The consequent logical concatenation of all the arcs pointing into a species leads to Boolean networks. For a Boolean representation of cellular interaction networks we propose a formalism based on logical (or signed) interaction hypergraphs, which facilitates in particular a logical steady state analysis (LSSA). LSSA enables studies on the logical processing of signals and the identification of optimal intervention points (targets) in cellular networks. LSSA also reveals network regions whose parametrization and initial states are crucial for the dynamic behavior.We have implemented these methods in our software tool CellNetAnalyzer (successor of FluxAnalyzer) and illustrate their applicability using a logical model of T-Cell receptor signaling providing non-intuitive results regarding feedback loops, essential elements, and (logical) signal processing upon different stimuli.ConclusionThe methods and formalisms we propose herein are another step towards the comprehensive functional analysis of cellular interaction networks. Their potential, shown on a realistic T-cell signaling model, makes them a promising tool.

A logical model provides insights into T cell receptor signaling

Cellular decisions are determined by complex molecular interaction networks. Large-scale signaling networks are currently being reconstructed, but the kinetic parameters and quantitative data that would allow for dynamic modeling are still scarce. Therefore, computational studies based upon the structure of these networks are of great interest. Here, a methodology relying on a logical formalism is applied to the functional analysis of the complex signaling network governing the activation of T cells via the T cell receptor, the CD4/CD8 co-receptors, and the accessory signaling receptor CD28. Our large-scale Boolean model, which comprises 94 nodes and 123 interactions and is based upon well-established qualitative knowledge from primary T cells, reveals important structural features (e.g., feedback loops and network-wide dependencies) and recapitulates the global behavior of this network for an array of published data on T cell activation in wild-type and knock-out conditions. More importantly, the model predicted unexpected signaling events after antibody-mediated perturbation of CD28 and after genetic knockout of the kinase Fyn that were subsequently experimentally validated. Finally, we show that the logical model reveals key elements and potential failure modes in network functioning and provides candidates for missing links. In summary, our large-scale logical model for T cell activation proved to be a promising in silico tool, and it inspires immunologists to ask new questions. We think that it holds valuable potential in foreseeing the effects of drugs and network modifications.

Non-T Cell Activation Linker (NTAL): A Transmembrane Adaptor Protein Involved in Immunoreceptor Signaling

A key molecule necessary for activation of T lymphocytes through their antigen-specific T cell receptor (TCR) is the transmembrane adaptor protein LAT (linker for activation of T cells). Upon TCR engagement, LAT becomes rapidly tyrosine phosphorylated and then serves as a scaffold organizing a multicomponent complex that is indispensable for induction of further downstream steps of the signaling cascade. Here we describe the identification and preliminary characterization of a novel transmembrane adaptor protein that is structurally and evolutionarily related to LAT and is expressed in B lymphocytes, natural killer (NK) cells, monocytes, and mast cells but not in resting T lymphocytes. This novel transmembrane adaptor protein, termed NTAL (non–T cell activation linker) is the product of a previously identified WBSCR5 gene of so far unknown function. NTAL becomes rapidly tyrosine-phosphorylated upon cross-linking of the B cell receptor (BCR) or of high-affinity Fcγ- and Fcɛ-receptors of myeloid cells and then associates with the cytoplasmic signaling molecules Grb2, Sos1, Gab1, and c-Cbl. NTAL expressed in the LAT-deficient T cell line J.CaM2.5 becomes tyrosine phosphorylated and rescues activation of Erk1/2 and minimal transient elevation of cytoplasmic calcium level upon TCR/CD3 cross-linking. Thus, NTAL appears to be a structural and possibly also functional homologue of LAT in non–T cells.

LIME A New Membrane Raft-associated Adaptor Protein Involved in CD4 and CD8 Coreceptor Signaling

Lymphocyte membrane rafts contain molecules critical for immunoreceptor signaling. Here, we report identification of a new raft-associated adaptor protein LIME (Lck-interacting molecule) expressed predominantly in T lymphocytes. LIME becomes tyrosine phosphorylated after cross-linking of the CD4 or CD8 coreceptors. Phospho-LIME associates with the Src family kinase Lck and its negative regulator, Csk. Ectopic expression of LIME in Jurkat T cells results in an increase of Csk in lipid rafts, increased phosphorylation of Lck and higher Ca2+ response to CD3 stimulation. Thus, LIME appears to be involved in regulation of T cell activation by coreceptors.

Regulation of in vitro and in vivo immune functions by the cytosolic adaptor protein SKAP-HOM.

ABSTRACT SKAP-HOM is a cytosolic adaptor protein representing a specific substrate for the Src family protein tyrosine kinase Fyn. Previously, several groups have provided experimental evidence that SKAP-HOM (most likely in cooperation with the cytosolic adaptor protein ADAP) is involved in regulating leukocyte adhesion. To further assess the physiological role of SKAP-HOM, we investigated the immune system of SKAP-HOM-deficient mice. Our data show that T-cell responses towards a variety of stimuli are unaffected in the absence of SKAP-HOM. Similarly, B-cell receptor (BCR)-mediated total tyrosine phosphorylation and phosphorylation of Erk, p38, and JNK, as well as immunoreceptor-mediated Ca2+ responses, are normal in SKAP-HOM−/− animals. However, despite apparently normal membrane-proximal signaling events, BCR-mediated proliferation is strongly attenuated in the absence of SKAP-HOM−/−. In addition, adhesion of activated B cells to fibronectin (a ligand for β1 integrins) as well as to ICAM-1 (a ligand for β2 integrins) is strongly reduced. In vivo, the loss of SKAP-HOM results in a less severe clinical course of experimental autoimmune encephalomyelitis following immunization of mice with the encephalitogenic peptide of MOG (myelin oligodendrocyte glycoprotein). This is accompanied by strongly reduced serum levels of MOG-specific antibodies and lower MOG-specific T-cell responses. In summary, our data suggest that SKAP-HOM is required for proper activation of the immune system, likely by regulating the cross-talk between immunoreceptors and integrins.

T cells and reactive oxygen species.

Reactive oxygen species (ROS) have been long considered simply as harmful by-products of metabolism, which damage cellular proteins, lipids, and nucleic acids. ROS are also known as a weapon of phagocytes, employed against pathogens invading the host. However, during the last decade, an understanding has emerged that ROS also have important roles as signaling messengers in a multitude of pathways, in all cells, tissues, and organs. T lymphocytes are the key players of the adaptive immune response, which both coordinate other immune cells and destroy malignant and virus-infected cells. ROS have been extensively implicated in T-cell hyporesponsiveness, apoptosis, and activation. It has also become evident that the source, the kinetics, and the localization of ROS production all influence cell responses. Thus, the characterization of the precise mechanisms by which ROS are involved in the regulation of T-cell functions is important for our understanding of the immune response and for the development of new therapeutic treatments against immune-mediated diseases. This review summarizes the 30-year-long history of research on ROS in T lymphocytes, with the emphasis on the physiological roles of ROS.

Transmembrane adapters: attractants for cytoplasmic effectors

Transmembrane adapter proteins (TRAPs) are a relatively new and growing family of proteins that include linker for activation of T cells (LAT), phosphoprotein associated with glycosphingolipid‐enriched micro domains (PAG)/C‐terminal Src kinase (Csk) binding protein (Cbp), SHP2‐interacting transmembrane adapter protein (SIT), T cell receptor interacting molecule (TRIM), and the recently identified non‐T cell activation linker (NTAL) and pp30. TRAPs share several common structural features, but more importantly they possess multiple sites of tyrosine phosphorylation, by which they act as scaffolds for recruiting cytosolic adapter and/or effector proteins. The membrane association of TRAPs places them near to the immunoreceptors, a position from which they coordinate and modulate the signals they receive to produce an appropriate cellular response.

Insulin and insulin-like growth factor-1 can modulate the phosphoinositide-3-kinase/Akt/FoxO1 pathway in SZ95 sebocytes in vitro

A recent hypothesis suggests that a high glycaemic load diet-associated increase of insulin-like growth factor-1 (IGF-1) and insulin may promote acne by reducing nuclear localization of the forkhead box-O1 (FoxO1) transcription factor via activation of the phosphoinositide-3-kinase (PI3K)/Akt pathway. Using SZ95 sebocytes as a model, we investigated the effect of the most important insulinotropic western dietary factors, IGF-1 and insulin on acne. SZ95 sebocytes were stimulated with different concentrations of IGF-1 and insulin (0.001, 0.01, 0.1 and 1 μM) for 15 to 120 min ± PI3K inhibitor LY294002 (50 μM). Cytoplasmic and nuclear protein expression of p-Akt and p-FoxO1 as well as FoxO transcriptional activity was analysed. In addition, the proliferation and differentiation of sebocytes and their TLR2/4 expression were determined. We found that high concentrations of IGF-1 and insulin differentially stimulate the PI3K/Akt/FoxO1 pathway by an early up-regulation of cytoplasmic p-Akt and delayed up-regulation of p-FoxO1 resulting in FoxO1 shift to the cytoplasm and the reduction of FoxO transcriptional activity, physiological serum concentration had no effect. IGF-1 at concentrations of 0.1 and 1 μM significantly reduced proliferation but increased differentiation of sebocytes to a greater extent than insulin (0.1 and 1 μM), but up-regulated TLR2/4 expression to comparable extent. These data provide the first in vitro evidence that FoxO1 principally might be involved in the regulation of growth-factor-stimulatory effects on sebaceous lipogenesis and inflammation in the pathological condition of acne. However, the in vivo significance under physiological conditions remains to be elucidated.

Structural and functional dissection of the cytoplasmic domain of the transmembrane adaptor protein SIT (SHP2-interacting transmembrane adaptor protein)

SIT (SHP2‐interacting transmembrane adaptor protein) is a recently identified transmembrane adaptor protein, which is expressed in lymphocytes. Its structural properties, in particular the presence of five potential tyrosine phosphorylation sites, suggest involvement of SIT in TCR‐mediated recruitment of SH2 domain‐containing intracellular signaling molecules to the plasma membrane. Indeed, it has recently been demonstrated that SIT inducibly interacts with the SH2‐containing protein tyrosine phosphatase 2 (SHP2) via an immunoreceptor tyrosine‐based inhibition motif (ITIM). Moreover, SIT is capable to inhibit TCR‐mediated signals proximal of activation of protein kinase C. However, inhibition of T cell activation by SIT occurs independently of SHP2 binding. The present study was performed to further characterize the molecular interaction between SIT and intracellular effector molecules and to identify the protein(s) mediating its inhibitory function. We demonstrate that SIT not only interacts with SHP2 but also with the adaptor protein Grb2 via two consensus YxN motifs. However, mutation of both Grb2‐binding sites also does not influence the inhibitory function of SIT. In contrast, mutation of the tyrosine‐based signaling motif Y168 ASV completely abrogates the ability of SIT to inhibit T cell activation. Co‐precipitation experiments revealed that the tyrosine kinase p50csk could represent the negative regulatory effector molecule that binds to this motif.

T Cell Activation Results in Conformational Changes in the Src Family Kinase Lck to Induce Its Activation

T cell activation involves the conformational activation of the tyrosine kinase Lck. Conformational Kinase Activation Lck is a tyrosine kinase that is critical for T cell activation, and its activity is induced by the T cell receptor (TCR). Phosphorylation of Lck at various residues either promotes or inhibits its activity, and Lck exists in various phosphorylated states in a T cell. With fluorescence lifetime imaging microscopic analysis of live human T cells and biochemical analyses, Stirnweiss et al. found that TCR activation produced a conformational change in Lck. In vitro studies showed that this “open” conformation of Lck exhibited enhanced kinase activity. Thus, phosphorylation, location, and conformation all potentially contribute to the regulation of Lck activity. The lymphocyte-specific Src family protein tyrosine kinase p56Lck (Lck) is essential for T cell development and activation and, hence, for adaptive immune responses. The mechanism by which Lck activity is directed toward specific substrates in response to T cell receptor (TCR) activation remains elusive. We used fluorescence lifetime imaging microscopy to assess the activation-dependent spatiotemporal changes in the conformation of Lck in live human T cells. Kinetic analysis of the fluorescence lifetime of Lck biosensors enabled the direct visualization of the dynamic local opening of 20% of the total amount of Lck proteins after activation of T cells with antibody against CD3 or by superantigen-loaded antigen-presenting cells. Parallel biochemical analysis of TCR complexes revealed that the conformational changes in Lck correlated with the induction of Lck enzymatic activity. These data show the dynamic, local activation through conformational change of Lck at sites of TCR engagement.