Large-scale network models of IL-1 and IL-6 signalling and their hepatocellular specification

The pro-inflammatory cytokines interleukin 1 (IL-1) and 6 (IL-6) are crucially involved in the regulation of a multitude of physiological processes, in particular coordinating the immune response upon bacterial infection and tissue injury. Both interleukins induce complex signalling cascades and trigger the production of mitogenic, pro-proliferative, anti-apoptotic, chemotactic, and pro-angiogenic factors thereby affecting the delicate balance between regeneration vs. invasive growth, tumourigenesis and metastasis. Moreover, several links to insulin resistance have been found within their associated signalling networks. Focusing on this from a systems biology perspective, we introduce comprehensive large-scale network models of IL-1 and IL-6 signalling which are based on a logical modelling approach and reflect the current biological knowledge. Theoretical network analysis enabled us to uncover general topological features and to make testable predictions on the stimulus-response behaviour of the networks. In this context, non-intuitive network-wide species dependencies as well as structures of regulatory feedback and feed-forward mechanisms could be characterised. By integrating high-throughput phosphoproteomic data from primary human hepatocytes we optimised the model structures to obtain models with high prediction accuracy for hepatocytes. Our model-based data analysis, for instance, suggested model modifications regarding (i) Akt contribution to IL-1-stimulated p38 MAPK activation and (ii) insignificant p38 MAPK activation in response to IL-6. In light of the presented results and in conjunction with the detailed model documentations, both models hold great potential for theoretical studies and practical applications.     

Binding of beta-D-glucopyranosyl bismethoxyphosphoramidate to glycogen phosphorylase b: kinetic and crystallographic studies

In an attempt to identify a new lead molecule that would enable the design of inhibitors with enhanced affinity for glycogen phosphorylase (GP), beta-D-glucopyranosyl bismethoxyphosphoramidate (phosphoramidate), a glucosyl phosphate analogue, was tested for inhibition of the enzyme. Kinetic experiments showed that the compound was a weak competitive inhibitor of rabbit muscle GPb (with respect to alpha-D-glucose-1-phosphate (Glc-1-P)) with a Ki value of 5.9 (+/-0.1) mM. In order to elucidate the structural basis of inhibition, we determined the structure of GPb complexed with the phosphoramidate at 1.83 A resolution. The complex structure reveals that the inhibitor binds at the catalytic site and induces significant conformational changes in the vicinity of this site. In particular, the 280s loop (residues 282-287) shifts 0.4-4.3 A (main-chain atoms) to accommodate the phosphoramidate, but these conformational changes do not lead to increased contacts between the inhibitor and the protein that would improve ligand binding.     

Carbon nanotube aqueous sol-gel composites: enzyme-friendly platforms for the development of stable biosensors

A new type of composite material based on carbon nanotubes and an aqueous sol-gel process has been developed. The electrochemical characteristics of these composites were investigated and compared to composites made with an alkoxy silane sol-gel process. The use of carbon nanotubes, as the conductive part of the composite, facilitated fast electron transfer rates. The feasibility of this type of composite for the development of biosensors was demonstrated using l-amino acid oxidase. The stability of the enzyme was increased when it was encapsulated in the aqueous sol-gel, and the sensor retained more that 50% of its response after 1 month of testing.     

Inhibition of phosphatidylinositol 3'-kinase induces preferentially killing of PTEN-null T leukemias through AKT pathway

We examined the functional role of the phosphatidylinositol 3'-kinase pathway in the growth and survival of cell lines of T-cell origin. Pharmacological inhibition of PI3'-kinase using LY294002 resulted in apoptosis of acute lymphoblastic T-cell leukemia (T-ALL) cell lines including CEM, Jurkat, and MOLT-4. On the other hand, the cutaneous T-cell lymphoma cell line HUT-78 was found to be refractory to LY294002- inducible apoptosis. Sensitivity or resistance to pharmacological inhibitors of PI3'-kinase correlated with tumor suppressor PTEN gene expression, as sensitive T-ALL cells do not express PTEN and have high level of activated AKT, in contrast to HUT-78 cells. Our data demonstrate that inhibition of PI3'-kinase results in dephosphorylation of AKT and partial inhibition of Bcl-xL expression in T-ALL cells, but not in HUT-78 cells. Interestingly, HUT-78 cells were also found to express higher levels of Bcl-xL protein as compared to T-ALL cells. Inhibition of PI3'-kinase also induces release of cytochrome c from mitochondria and activation of caspase-3 and PARP in all T-ALL cell lines tested, but not in HUT-78 cells. Taken altogether, our data demonstrate that the PI3'-kinase/AKT pathway plays a major role in the growth and survival of PTEN-null T-ALL cells, and identify this cascade as promising target for therapeutic intervention in acute T-cell leukemias.     

alpha-synuclein is required for the fibrillar nature of ubiquitinated inclusions induced by proteasomal inhibition in primary neurons

Proteasomal dysfunction may underlie certain neuro-degenerative conditions such as Parkinson disease. We have shown that pharmacological inhibition of the proteasome in cultured neuronal cells leads to apoptotic death and formation of cytoplasmic ubiquitinated inclusions. These inclusions stain for alpha-synuclein and assume a fibrillar structure, as assessed by thioflavine S staining, and therefore resemble Lewy bodies. alpha-Synuclein is thought to be a central component of Lewy bodies. Whether alpha-synuclein is required for inclusion formation or apoptotic death has not been formally assessed. The present study examines whether alpha-synuclein deficiency in neurons alters their sensitivity to proteasomal inhibition-induced apoptosis or inclusion formation. Cortical neurons derived from alpha-synuclein-null mice showed a similar sensitivity to death induced by the proteasomal inhibitor lactacystin compared with neurons derived from wild-type mice. Furthermore, the absence of alpha-synuclein did not influence the percentage of lactacystin-treated neurons harboring cytoplasmic ubiquitinated inclusions or alter the solubility of such inclusions. In contrast, however, ubiquitinated inclusions in alpha-synuclein-deficient neurons lacked amyloid-like fibrillization, as determined by thioflavine S staining. This indicates that although alpha-synuclein deficiency does not affect the formation of ubiquitinated inclusions, it does significantly alter their structure. The lack of effect on survival in alpha-synuclein knock-out cultures further suggests that the fibrillar nature of the inclusions does not contribute to neuronal degeneration in this model.     

Interactions between Lipids and Human Anti-HIV Antibody 4E10 Can Be Reduced without Ablating Neutralizing Activity

Human 4E10 is one of the broadest-specificity, HIV-1-neutralizing monoclonal antibodies known, recognizing a membrane-proximal linear epitope on gp41. The lipid cross-reactivity of 4E10 has been alternately suggested either to contribute to the apparent rarity of 4E10-like antibody responses in HIV infections, through elimination by B-cell tolerance mechanisms to self-antigens, or to contribute to neutralization potency by virus-specific membrane binding outside of the membrane-proximal external region (MPER). To investigate how 4E10 interacts with membrane and protein components, and whether such interactions contribute to neutralization mechanisms, we introduced two mutations into 4E10 Fv constructs, Trp to Ala at position 100 in the heavy chain [W(H100)A] and Gly to Glu at position 50 in the light chain [G(L50)E], selected to disrupt potential lipid interactions via different mechanisms. Wild-type and mutant Fvs all bound with the same affinity to peptides and monomeric and trimeric gp140s, but the affinities for gp140s were uniformly 10-fold weaker than to peptides. 4E10 Fv binding responses to liposomes in the presence or absence of MPER peptides were weak in absolute terms, consistent with prior observations, and both mutations attenuated interactions even further, as predicted. The W(H100)A mutation reduced neutralization efficiency against four HIV-1 isolates, but the G(L50)E mutation increased potency across the same panel. Electron paramagnetic resonance experiments showed that the W(H100)A mutation, but not the G(L50)E mutation, reduced the ability of 4E10 to extract MPER peptides from membranes. These results show that 4E10 nonspecific membrane binding is separable from neutralization, which is achieved through specific peptide/lipid orientation changes.Few of the hundreds of known neutralizing anti-HIV monoclonal antibodies (MAbs) display broad cross-reactive activities (4). Of those derived from clade B-infected patients, b12 binds to the gp120 subunit of the HIV envelope protein (Env), to an epitope that overlaps the CD4 binding site, and neutralizes approximately 50% of virus isolates tested, including non-clade B viruses (27). 2G12 binds to N-linked carbohydrates on gp120 (32, 34) and neutralizes 41% of isolates tested, although not clade C or E isolates. 447-52D also binds to the gp120 subunit, to an epitope within the V3 loop, and potently neutralizes up to 45% of clade B isolates but rarely non-clade B isolates. 4E10 and 2F5 recognize adjacent epitopes located at the membrane-proximal external region (MPER) of the gp41 Env subunit (9, 22, 24, 28, 42). Two neutralizing antibodies (NAbs) isolated from a clade A-infected patient (PG9 and PG16) show broad and potent neutralizing activity by recognizing epitopes consisting of conserved regions of the V2 and V3 loops of gp120, preferentially on native trimers (40).4E10 is capable of neutralizing all isolates tested at some level (4), although there is evidence for the existence of rare viruses that are resistant to 4E10 neutralization (30). The exact structure of the epitope recognized by 4E10 within the trimeric, functional HIV Env is unknown, but structural studies have shown that an isolated peptide spanning the epitope adopts a helical conformation, a short 3₁₀ segment followed by a 4₁₃ (or true α-helical) segment, with an extended structure at the N terminus when bound to 4E10 (9). It has also been reported that 4E10 interacts with a variety of lipids and membrane components, particularly the phospholipid cardiolipin (15), suggesting that difficulties in eliciting 4E10-like broadly neutralizing antibodies by immunization and the apparent rarity of 4E10-like antibody responses in HIV-1-infected subjects (19, 33) are linked to this polyspecificity to autoantigens, contributing to their elimination through tolerance mechanisms. However, subsequent studies have shown that the measurable, but quite weak, affinity of 4E10 for certain lipids is comparable to that of some antiphospholipid antibodies elicited during many infections, suggesting that 4E10 is not remarkably autoreactive (35). Therefore, it is still unclear whether lipid binding properties are linked to the rarity of 4E10-like specificities. It has also been proposed that the neutralizing activity of 4E10 may partly depend on lipid binding, either through interactions with viral membrane lipids that disturb the membrane-bound structure of the MPER on the trimeric, virion-associated Env spike (39) or through an encounter model. In the latter, initial interactions with membrane components align 4E10 with its protein epitope or allow 4E10 to gain proximity to its epitope (1), perhaps partially alleviating steric occlusion effects (for example, see reference 17). We sought to determine whether specific interactions exist between 4E10 and membrane lipid components and whether such interactions meaningfully contribute to neutralization by any mechanism.