Characterization of the structure of RAMP1 by mutagenesis and molecular modeling

Receptor activity modifying proteins (RAMPs) are a family of single-pass transmembrane proteins that dimerize with G-protein-coupled receptors. They may alter the ligand recognition properties of the receptors (particularly for the calcitonin receptor-like receptor, CLR). Very little structural information is available about RAMPs. Here, an ab initio model has been generated for the extracellular domain of RAMP1. The disulfide bond arrangement (Cys 27-Cys82, Cys40-Cys72, and Cys 57-Cys104) was determined by site-directed mutagenesis. The secondary structure (a-helices from residues 29-51, 60-80, and 87-100) was established from a consensus of predictive routines. Using these constraints, an assemblage of 25,000 structures was constructed and these were ranked using an all-atom statistical potential. The best 1000 conformations were energy minimized. The lowest scoring model was refined by molecular dynamics simulation. To validate our strategy, the same methods were applied to three proteins of known structure; PDB:1HP8, PDB:1V54 chain H (residues 21-85), and PDB:1T0P. When compared to the crystal structures, the models had root mean-square deviations of 3.8 Å, 4.1 Å, and 4.0 Å, respectively. The model of RAMP1 suggested that Phe93, Tyr 100, and Phe101 form a binding interface for CLR, whereas Trp74 and Phe92 may interact with ligands that bind to the CLR/RAMP1 heterodimer. © 2006 by the Biophysical Society.

Identifying a novel functional domain within the p115 tethering factor required for Golgi ribbon assembly and membrane trafficking

The tethering factor p115 has been shown to facilitate Golgi biogenesis and membrane traffic in cells in culture. However, the role of p115 within an intact animal is largely unknown. Here, we document that RNAi-mediated depletion of p115 in C. elegans causes accumulation of the yolk protein (YP170) in body cavity and the retention of the yolk receptor RME-2 in the ER and the Golgi within oocytes.Structure-function analyses of p115 have identified two homology (H1-2) regions within the N-terminal globular head and the coiled-coil 1 (CC1) domain as essential for p115 function. We identify a novel C-terminal domain of p115 as necessary for Golgi ribbon formation and cargo trafficking. We show that p115 mutants lacking the fourth CC domain (CC4) act in a dominant negative manner to disrupt Golgi and prevent cargo trafficking in cells containing endogenous p115. Furthermore, using RNAi-mediated "replacement" strategy we show that CC4 is necessary for Golgi ribbon formation and membrane trafficking in cells depleted of endogenous p115.p115 has been shown to bind a subset of ER-Golgi SNAREs through CC1 and CC4 domains (Shorter et al., 2002). Our findings show that CC4 is required for p115 function and suggest that both the CC1 and the CC4 SNARE-binding motifs may participate in p115-mediated membrane tethering.

Development of a probabilistic graphical structure from a model of mental health clinical expertise

This thesis explores the process of developing a principled approach for translating a model of mental-health risk expertise into a probabilistic graphical structure. Probabilistic graphical structures can be a combination of graph and probability theory that provide numerous advantages when it comes to the representation of domains involving uncertainty, domains such as the mental health domain. In this thesis the advantages that probabilistic graphical structures offer in representing such domains is built on. The Galatean Risk Screening Tool (GRiST) is a psychological model for mental health risk assessment based on fuzzy sets. In this thesis the knowledge encapsulated in the psychological model was used to develop the structure of the probability graph by exploiting the semantics of the clinical expertise. This thesis describes how a chain graph can be developed from the psychological model to provide a probabilistic evaluation of risk that complements the one generated by GRiST’s clinical expertise by the decomposing of the GRiST knowledge structure in component parts, which were in turned mapped into equivalent probabilistic graphical structures such as Bayesian Belief Nets and Markov Random Fields to produce a composite chain graph that provides a probabilistic classification of risk expertise to complement the expert clinical judgements

In silico modelling of the interaction of flavonoids with human P-glycoprotein nucleotide-binding domain

A three-dimensional model of human ABCB1 nucleotide-binding domain (NBD) was developed by homology modelling using the high-resolution human TAP1 transporter structure as template. Interactions between NBD and flavonoids were investigated using in silico docking studies. Ring-A of unmodified flavonoid was located within the NBD P-loop with the 5-hydroxyl group involved in hydrogen bonding with Lys1076. Ring-B was stabilised by hydrophobic stacking interactions with Tyr1044. The 3-hydroxyl group and carbonyl oxygen were extensively involved in hydrogen bonding interactions with amino acids within the NBD. Addition of prenyl, benzyl or geranyl moieties to ring-A (position-6) and hydrocarbon substituents (O-n-butyl to O-n-decyl) to ring-B (position-4) resulted in a size-dependent decrease in predicted docking energy which reflected the increased binding affinities reported in vitro.

Category structure affects the developmental trajectory of children’s inductive inferences for both natural kinds and artefacts

Inductive reasoning is fundamental to human cognition, yet it remains unclear how we develop this ability and what might influence our inductive choices. We created novel categories in which crucial factors such as domain and category structure were manipulated orthogonally. We trained 403 4-9-year-old children to categorise well-matched natural kind and artefact stimuli with either featural or relational category structure, followed by induction tasks. This wide age range allowed for the first full exploration of the developmental trajectory of inductive reasoning in both domains. We found a gradual transition from perceptual to categorical induction with age. This pattern was stable across domains, but interestingly, children showed a category bias one year later for relational categories. We hypothesise that the ability to use category information in inductive reasoning develops gradually, but is delayed when children need to process and apply more complex category structures. © 2014 © 2014 Taylor & Francis.

ERK5:structure, regulation and function

Extracellular signal-regulated kinase 5 (ERK5), also termed big mitogen-activated protein kinase-1 (BMK1), is the most recently identified member of the mitogen-activated protein kinase (MAPK) family and consists of an amino-terminal kinase domain, with a relatively large carboxy-terminal of unique structure and function that makes it distinct from other MAPK members. It is ubiquitously expressed in numerous tissues and is activated by a variety of extracellular stimuli, such as cellular stresses and growth factors, to regulate processes such as cell proliferation and differentiation. Targeted deletion of Erk5 in mice has revealed that the ERK5 signalling cascade plays a critical role in cardiovascular development and vascular integrity. Recent data points to a potential role in pathological conditions such as cancer and tumour angiogenesis. This review focuses on the physiological and pathological role of ERK5, the regulation of this kinase and the recent development of small molecule inhibitors of the ERK5 signalling cascade. © 2012 Elsevier Inc.