Benjamin A. Hall

Structural Flexibility of the Macrophage Dengue Virus Receptor CLEC5A: IMPLICATIONS FOR LIGAND BINDING AND SIGNALING*

The human C-type lectin-like molecule CLEC5A is a critical macrophage receptor for dengue virus. The binding of dengue virus to CLEC5A triggers signaling through the associated adapter molecule DAP12, stimulating proinflammatory cytokine release. We have crystallized an informative ensemble of CLEC5A structural conformers at 1.9-Å resolution and demonstrate how an on-off extension to a β-sheet acts as a binary switch regulating the flexibility of the molecule. This structural information together with molecular dynamics simulations suggests a mechanism whereby extracellular events may be transmitted through the membrane and influence DAP12 signaling. We demonstrate that CLEC5A is homodimeric at the cell surface and binds to dengue virus serotypes 1–4. We used blotting experiments, surface analyses, glycan microarray, and docking studies to investigate the ligand binding potential of CLEC5A with particular respect to dengue virus. This study provides a rational foundation for understanding the dengue virus-macrophage interaction and the role of CLEC5A in dengue virus-induced lethal disease.

In vivo tau PET imaging in dementia: Pathophysiology, radiotracer quantification, and a systematic review of clinical findings.

In addition to the deposition of β-amyloid plaques, neurofibrillary tangles composed of aggregated hyperphosphorylated tau are one of the pathological hallmarks of Alzheimers disease and other neurodegenerative disorders. Until now, our understanding about the natural history and topography of tau deposition has only been based on post-mortem and cerebrospinal fluid studies, and evidence continues to implicate tau as a central driver of downstream neurodegenerative processes and cognitive decline. Recently, it has become possible to assess the regional distribution and severity of tau burden in vivo with the development of novel radiotracers for positron emission tomography (PET) imaging. In this article, we provide a comprehensive discussion of tau pathophysiology, its quantification with novel PET radiotracers, as well as a systematic review of tau PET imaging in normal aging and various dementia conditions: mild cognitive impairment, Alzheimers disease, frontotemporal dementia, progressive supranuclear palsy, and Lewy body dementia. We discuss the main findings in relation to group differences, clinical-cognitive correlations of tau PET, and multi-modal relationships among tau PET and other pathological markers. Collectively, the small but growing literature of tau PET has yielded consistent anatomical patterns of tau accumulation that recapitulate post-mortem distribution of neurofibrillary tangles which correlate with cognitive functions and other markers of pathology. In general, AD is characterised by increased tracer retention in the inferior temporal lobe, extending into the frontal and parietal regions in more severe cases. It is also noted that the spatial topography of tau accumulation is markedly distinct to that of amyloid burden in aging and AD. Tau PET imaging has also revealed characteristic spatial patterns among various non-AD tauopathies, supporting its potential role for differential diagnosis. Finally, we propose novel directions for future tau research, including (a) longitudinal imaging in preclinical dementia, (b) multi-modal mapping of tau pathology onto other pathological processes such as neuroinflammation, and (c) the need for more validation studies against post-mortem samples of the same subjects.

Probing the solution structure of IκB kinase (IKK) subunit γ and its interaction with Kaposi sarcoma-associated herpes virus Flice-interacting protein and IKK subunit β by EPR spectroscopy.

Viral flice-interacting protein (vFLIP), encoded by the oncogenic Kaposi sarcoma-associated herpes virus (KSHV), constitutively activates the canonical nuclear factor κ-light-chain-enhancer of activated B cells (NF-κB) pathway. This is achieved through subversion of the IκB kinase (IKK) complex (or signalosome), which involves a physical interaction between vFLIP and the modulatory subunit IKKγ. Although this interaction has been examined both in vivo and in vitro, the mechanism by which vFLIP activates the kinase remains to be determined. Because IKKγ functions as a scaffold, recruiting both vFLIP and the IKKα/β subunits, it has been proposed that binding of vFLIP could trigger a structural rearrangement in IKKγ conducive to activation. To investigate this hypothesis we engineered a series of mutants along the length of the IKKγ molecule that could be individually modified with nitroxide spin labels. Subsequent distance measurements using electron paramagnetic resonance spectroscopy combined with molecular modeling and molecular dynamics simulations revealed that IKKγ is a parallel coiled-coil whose response to binding of vFLIP or IKKβ is localized twisting/stiffening and not large-scale rearrangements. The coiled-coil comprises N- and C-terminal regions with distinct registers accommodated by a twist: this structural motif is exploited by vFLIP, allowing it to bind and subsequently activate the NF-κB pathway. In vivo assays confirm that NF-κB activation by vFLIP only requires the N-terminal region up to the transition between the registers, which is located directly C-terminal of the vFLIP binding site.

Bringing LTL Model Checking to Biologists

The BioModelAnalyzer (BMA) is a web based tool for the development of discrete models of biological systems. Through a graphical user interface, it allows rapid development of complex models of gene and protein interaction networks and stability analysis without requiring users to be proficient computer programmers. Whilst stability is a useful specification for testing many systems, testing temporal specifications in BMA presently requires the user to perform simulations. Here we describe the LTL module, which includes a graphical and natural language interfaces to testing LTL queries. The graphical interface allows for graphical construction of the queries and presents results visually in keeping with the current style of BMA. The Natural language interface complements the graphical interface by allowing a gentler introduction to formal logic and exposing educational resources.

In vivo cross-linking and transmembrane helix dynamics support a non-piston model of signaling within E. coli EnvZ

In gram-negative bacteria, porins span the outer membrane and control the influx of several prominent groups of antibiotics. Thus, it should not be surprising that expression of these porins is often altered in clinical isolates that exhibit multidrug resistance (MDR). The major regulator of porin expression in Escherichia coli is EnvZ, a canonical sensor histidine kinase (SHK). It allosterically processes periplasmic interactions with MzrA and cytoplasmic osmosensing into a single unified change in the ratio of its kinase and phosphatase activities. Unfortunately, the role of the transmembrane domain (TMD) in communicating these signals across the cellular membrane remains not well understood. Here, we employed in vivo sulfhydryl-reactivity to probe the dynamics of individual TM2 helices within the TMD and demonstrate that upon stimulus perception, EnvZ employs a non-piston-type mechanism of transmembrane communication. In silico coarse-grained molecular dynamics (CG-MD) simulations with EnvZ TM2 are in agreement with these in vivo results. We conclude by discussing these results within the context of allosteric processing by EnvZ and propose that these results can be used to predict and classify transmembrane communication by various SHKs.

Tumor pre-conditioning of draining lymph node stroma by lactic acid

Communication between tumors and the stroma of tumor draining lymph nodes (TDLNs) exists before metastasis arises, altering structure and function of the TDLN niche. Transcriptional profiling of fibroblastic reticular cells (FRCs), the dominant stromal population of the LN, revealed reprogramming of these cells in immune related pathways, but also in fibroblast activation and mitochondrial function. However, tumor derived factors driving the changes in FRCs remained to be identified. Taking an unbiased approach, we show that lactate, a metabolite released by cancer cells, elicits upregulation of Pdpn and Thy1 in FRCs of TDLNs, making them akin to activated fibroblasts found at the primary tumor site. Furthermore, we show that tumor-derived lactate alters mitochondrial function of FRCs of TDLNs. Thus, our results demonstrate a novel mechanism by which a tumor-derived metabolite modulates the function of fibroblasts in TDLNs.

Exploring the role of stromal osmoregulation in cancer and disease using executable modelling

Osmotic regulation is a vital homoeostatic process in all cells and tissues. Cells initially respond to osmotic stresses by activating transmembrane transport proteins to move osmotically active ions. Disruption of ion and water transport is frequently observed in cellular transformations such as cancer. We report that genes involved in membrane transport are significantly deregulated in many cancers, and that their expression can distinguish cancer cells from normal cells with a high degree of accuracy. We present an executable model of osmotic regulation and membrane transport in mammalian cells, providing a mechanistic explanation for phenotype change in varied disease states, and accurately predicting behaviour from single cell expression data. We also predict key proteins involved in cellular transformation, SLC4A3 (AE3), and SLC9A1 (NHE1). Furthermore, we predict and verify a synergistic drug combination in vitro, of sodium and chloride channel inhibitors, which target the osmoregulatory network to reduce cancer-associated phenotypes in fibroblasts.Aberrant ion transporter expression disrupts osmoregulation in many cancers. Here, the authors introduce an executable model of osmotic regulation and membrane transport, illuminating the mechanistic basis of multiple cellular cancer phenotypes and suggesting therapeutic avenues.

Linear Temporal Logic for Biologists in BMA

This is the author accepted manuscript. The final version is available from Springer via http://www.springer.com/in/book/9783319451763

Deregulation of Osmotic Regulation Machinery Explains and Predicts Cellular Transformation in Cancer and Disease

Osmotic regulation is a hugely important homeostatic system in all cells. Cells respond to osmotic stresses by activating or upregulating proteins involved in the transportation of charged ions, primarily Chlorine, Potassium, Sodium, and Calcium. Additionally, the movement of ions and osmotically obliged water are necessary for many of the cellular hallmarks exhibited in the transformations associated with disease states such as cancer. In particular, the aberrant expression of ion channels are hallmarks for increased proliferative and invasive behaviours ( [1, 2]). We present a formal model of the osmotic regulation machinery within a mammalian cell. The model can provide a mechanistic explanation for the behavioural changes observed in highly diverse cellular systems of murine premetastatic Lymph Node stromal cells, and Lung Cancer Fibroblasts. The model explains phenotypic transformations within each cell types, and predicts behaviour from datasets not involved in its generation. Furthermore, we use the model to predict key proteins involved in each transformation, and propose experiments to alter the behaviour of cells in controllable ways.