Molecular evolution of CXCR1, a G protein-coupled receptor involved in signal transduction of neutrophils

Human neutrophils are a type of white blood cell, which forms an early line of defense against bacterial infections. Neutrophils are highly responsive to the chemokine, interleukin-8 (IL-8) due to the abundant distribution of CXCR1, one of the IL-8 receptors on the neutrophil cell surface. As a member of the GPCR family, CXCR1 plays a crucial role in the IL-8 signal transduction pathway in neutrophils. We sequenced the complete coding region of the CXCR1 gene in worldwide human populations and five representative nonhuman primate species. Our results indicate accelerated protein evolution in the human lineage, which was likely caused by Darwinian positive selection. The sliding window analysis and the codon-based neutrality test identified signatures of positive selection at the N-terminal ligand/receptor recognition domain of human CXCR1.

Robustness and dissipation of mitogen-activated protein kinases signal transduction network: Underlying funneled landscape against stochastic fluctuations

We uncovered the underlying energy landscape of the mitogen-activated protein kinases signal transduction cellular network by exploring the statistical natures of the Brownian dynamical trajectories. We introduce a dimensionless quantity: The robustness ratio of energy gap versus local roughness to measure the global topography of the underlying landscape. A high robustness ratio implies funneled landscape. The landscape is quite robust against environmental fluctuations and variants of the intrinsic chemical reaction rates.

Funneled landscape leads to robustness of cellular networks: MAPK signal transduction

We uncover the underlying potential energy landscape for a cellular network. We find that the potential energy landscape of the mitogen-activated protein-kinase signal transduction network is funneled toward the global minimum. The funneled landscape is quite robust against random perturbations. This naturally explains robustness from a physical point of view. The ratio of slope versus roughness of the landscape becomes a quantitative measure of robustness of the network. Funneled landscape is a realization of the Darwinian principle of natural selection at the cellular network level. It provides an optimal criterion for network connections and design. Our approach is general and can be applied to other cellular networks.

Akt signal transduction dysfunction in the 3xTg-AD mouse model of Alzheimer's disease

Alzheimer’s disease (AD) is an incurable neurodegenerative disorder, accounting for over 60% of all cases of dementia. The primary risk factor for AD is age, however several genetic and environmental factors are also involved. The pathological characteristics of AD include extracellular deposition of the beta-amyloid peptide (Aβ) and intraneuronal accumulation of neurofibrillary tangles (NFTs) made of aggregated paired helical filaments (PHFs) of the hyperphosphorylated tau protein, along with synaptic loss and neuronal death. There are numerous biochemical mechanisms involved in AD pathogenesis, however the reigning hypothesis points to toxic oligomeric Aβ species as the primary causative factor in a cascade of events leading to neuronal stress and dyshomeostasis that initiate abnormal regulation of tau. The insulin and IGF-1 receptors (IR, IGF-1R) are the primary activators of PI3- K/Akt through which they regulate cell growth, development, glucose metabolism, and learning and memory. Work in our lab and others shows increased Akt activity and phosphorylation of its downstream targets in AD brain, along with insulin and insulin-like growth factor-1 signalling (IIS) dysfunction. This is supported by studies of AD models in vivo and in vitro. Our group and others hypothesise that Aβ activates Akt through IIS to initiate a negative feedback mechanism that desensitises neurons to insulin/IGF-1, and sustains activation of Akt. In this study the functions of endogenous Akt, IR, and the insulin receptor substrate (IRS-1) were examined in relationship to Aβ and tau pathology in the 3xTg-AD mouse model, which contains three mutant human transgenes associated with familial AD or dementia. The 3xTg-AD mouse develops Aβ and tau pathology in a spatiotemporal manner that best recapitulates the progression of AD in human brain. Western blotting and immunofluorescent microscopy techniques were utilised in vivo and in vitro, to examine the relationship between IIS, Akt, and AD pathology. I first characterised in detail AD pathology in 3xTg-AD mice, where an age-related accumulation of intraneuronal Aβ and tau was observed in the hippocampal formation, amygdala, and entorhinal cortex, and at late stages (18 months), extracellular amyloid plaques and NFTs, primarily in the subiculum and the CA1 layer of the hippocampal formation. Increased activity of Akt, detected with antibody to phosphoSer473-Akt, was increased in 3xTg-AD mice compared to age-matched non-transgenic mice (non-Tg), and in direct correlation to the accumulation of Aβ and tau in neuronal somatodendritic compartments. Akt phosphorylates tau at residue Ser214 within a highly specific consensus sequence for Akt phosphorylation, and phosphoSer214-tau strongly decreases microtubule (MT) stabilisation by preventing tau-MT binding. PhosphoSer214-tau increased concomitantly with this in the same age-related and region-specific fashion. Polarisation of tau phosphorylation was observed, where PHF-1 (tauSer396/404) and phosphoSer214-tau both appeared early in 3xTg-AD mice in distinct neuronal compartments: PHF-1 in axons, and phosphoSer214-tau in neuronal soma and dendrites. At 18 months, phosphoSer214-tau strongly colocalised with NFTs positive for the PHF- 1 and AT8 (tauSer202/Thr205) phosphoepitopes. IR was decreased with age in 3xTg-AD brain and in comparison to age-matched non-Tg, and this was specific for brain regions containing Aβ, tau, and hyperactive Akt. IRS-1 was similarly decreased, and both proteins showed altered subcellular distribution. Phosphorylation of IRS-1Ser312 is a strong indicator of IIS dysfunction and insulin resistance, and was increased in 3xTg-AD mice with age and in relation to pathology. Of particular note was our observation that abberant IIS and Akt signalling in 3xTg-AD brain related to Aβ and tau pathology on a gross anatomical level, and specifically localised to the brain regions and circuitry of the perforant path. Finally, I conducted a preliminary study of the effects of synthetic Aβ oligomers on embryonic rat hippocampus neuronal cultures to support these results and those in the literature. Taken together, these novel findings provide evidence for IIS and Akt signal transduction dysfunction as the missing link between Aβ and tau pathogenesis, and contribute to the overall understanding of the biochemical mechanisms of AD.

Smoothened signal transduction is promoted by G protein-coupled receptor kinase 2.

Deregulation of the Sonic hedgehog pathway has been implicated in an increasing number of human cancers. In this pathway, the seven-transmembrane (7TM) signaling protein Smoothened regulates cellular proliferation and differentiation through activation of the transcription factor Gli. The activity of mammalian Smoothened is controlled by three different hedgehog proteins, Indian, Desert, and Sonic hedgehog, through their interaction with the Smoothened inhibitor Patched. However, the mechanisms of signal transduction from Smoothened are poorly understood. We show that a kinase which regulates signaling by many "conventional" 7TM G-protein-coupled receptors, G protein-coupled receptor kinase 2 (GRK2), participates in Smoothened signaling. Expression of GRK2, but not catalytically inactive GRK2, synergizes with active Smoothened to mediate Gli-dependent transcription. Moreover, knockdown of endogenous GRK2 by short hairpin RNA (shRNA) significantly reduces signaling in response to the Smoothened agonist SAG and also inhibits signaling induced by an oncogenic Smoothened mutant, Smo M2. We find that GRK2 promotes the association between active Smoothened and beta-arrestin 2. Indeed, Gli-dependent signaling, mediated by coexpression of Smoothened and GRK2, is diminished by beta-arrestin 2 knockdown with shRNA. Together, these data suggest that GRK2 plays a positive role in Smoothened signaling, at least in part, through the promotion of an association between beta-arrestin 2 and Smoothened.

Adrenergic receptors. Models for regulation of signal transduction processes.

Adrenergic receptors are prototypic models for the study of the relations between structure and function of G protein-coupled receptors. Each receptor is encoded by a distinct gene. These receptors are integral membrane proteins with several striking structural features. They consist of a single subunit containing seven stretches of 20-28 hydrophobic amino acids that represent potential membrane-spanning alpha-helixes. Many of these receptors share considerable amino acid sequence homology, particularly in the transmembrane domains. All of these macromolecules share other similarities that include one or more potential sites of extracellular N-linked glycosylation near the amino terminus and several potential sites of regulatory phosphorylation that are located intracellularly. By using a variety of techniques, it has been demonstrated that various regions of the receptor molecules are critical for different receptor functions. The seven transmembrane regions of the receptors appear to form a ligand-binding pocket. Cysteine residues in the extracellular domains may stabilize the ligand-binding pocket by participating in disulfide bonds. The cytoplasmic domains contain regions capable of interacting with G proteins and various kinases and are therefore important in such processes as signal transduction, receptor-G protein coupling, receptor sequestration, and down-regulation. Finally, regions of these macromolecules may undergo posttranslational modifications important in the regulation of receptor function. Our understanding of these complex relations is constantly evolving and much work remains to be done. Greater understanding of the basic mechanisms involved in G protein-coupled, receptor-mediated signal transduction may provide leads into the nature of certain pathophysiological states.

Multivariate Statistical Analysis Applied to an IL6 Signal Transduction Model in Hepatocytes

This paper introduces the application of linear multivariate statistical techniques, including partial least squares (PLS), canonical correlation analysis (CCA) and reduced rank regression (RRR), into the area of Systems Biology. This new approach aims to extract the important proteins embedded in complex signal transduction pathway models.The analysis is performed on a model of intracellular signalling along the janus-associated kinases/signal transducers and transcription factors (JAK/STAT) and mitogen activated protein kinases (MAPK) signal transduction pathways in interleukin-6 (IL6) stimulated hepatocytes, which produce signal transducer and activator of transcription factor 3 (STAT3).A region of redundancy within the MAPK pathway that does not affect the STAT3 transcription was identified using CCA. This is the core finding of this analysis and cannot be obtained by inspecting the model by eye. In addition, RRR was found to isolate terms that do not significantly contribute to changes in protein concentrations, while the application of PLS does not provide such a detailed picture by virtue of its construction.This analysis has a similar objective to conventional model reduction techniques with the advantage of maintaining the meaning of the states prior to and after the reduction process. A significant model reduction is performed, with a marginal loss in accuracy, offering a more concise model while maintaining the main influencing factors on the STAT3 transcription.The findings offer a deeper understanding of the reaction terms involved, confirm the relevance of several proteins to the production of Acute Phase Proteins and complement existing findings regarding cross-talk between the two signalling pathways.

Homodimerization Is Essential for the Receptor for Advanced Glycation End Products (RAGE)-mediated Signal Transduction

The receptor for advanced glycation end products (RAGE) is a pattern-recognition receptor that binds to diverse ligands and initiates a downstream proinflammatory signaling cascade. RAGE activation has been linked to diabetic complications, Alzheimer disease, infections, and cancers. RAGE is known to mediate cell signaling and downstream proinflammatory gene transcription activation, although the precise mechanism surrounding receptor-ligand interactions is still being elucidated. Recent fluorescence resonance energy transfer evidence indicates that RAGE may form oligomers on the cell surface and that this could be related to signal transduction. To investigate whether RAGE forms oligomers, protein-protein interaction assays were carried out. Here, we demonstrate the interaction between RAGE molecules via their N-terminal V domain, which is an important region involved in ligand recognition. By protein cross-linking using water-soluble and membrane-impermeable cross-linker bis(sulfosuccinimidyl) suberate and nondenaturing gels, we show that RAGE forms homodimers at the plasma membrane, a process potentiated by S100B and advanced glycation end products. Soluble RAGE, the RAGE inhibitor, is also capable of binding to RAGE, similar to V peptide, as shown by surface plasmon resonance. Incubation of cells with soluble RAGE or RAGE V domain peptide inhibits RAGE dimerization, subsequent phosphorylation of intracellular MAPK proteins, and activation of NF-kappa B pathways. Thus, the data indicate that dimerization of RAGE represents an important component of RAGE-mediated cell signaling.