GPCRs

With our increasing appreciation of the true complexity of diseases and pathophysiologies, it is clear that this knowledge needs to inform the future development of pharmacotherapeutics. For many disorders, the disease mechanism itself is a complex process spanning multiple signaling networks, tissues, and organ systems. Identifying the precise nature and locations of the pathophysiology is crucial for the creation of systemically effective drugs. Diseases once considered constrained to a limited range of organ systems, e.g., central neurodegenerative disorders such as Alzheimer's disease (AD), Parkinson's disease (PD), and Huntington' disease (HD), the role of multiple central and peripheral organ systems in the etiology of such diseases is now widely accepted. With this knowledge, it is increasingly clear that these seemingly distinct neurodegenerative disorders (AD, PD, and HD) possess multiple pathophysiological similarities thereby demonstrating an inter-related con...

Video of my talk at MIT, Center for Bits and Atoms (Andreas Mershin's lab), 9 December 2015: https://www.youtube.com/watch?v=ej45lyl3rzA

Research on olfaction is on the rise. The discovery of the olfactory receptor genes by Linda Buck and Richard Axel in 1991 catapulted olfaction from eccentricity into core neurobiological research. With the identification of the receptors as G-protein coupled receptors olfactory research became affiliated with a wide range of cell signaling studies. While a lot of fundamental questions about olfactory processing are still open to dispute, the consensus is that the olfactory pathway will facilitate a greater neurobiological understanding of signal processing. One of the open questions that attracted greater attention over the past ten years is the longstanding problem of how the brain ‘maps’ smells. My postdoc project centers on olfaction to analyze theoretical and experimental breakthroughs through scientific methodology. Its current dynamics and susceptibility to the revision of its core premises makes olfactory research an excellent example to study the ambiguity of determining what is a reliable research strategy. One of the key reasons for the historical marginalization of olfaction was the experimental difficulty of conducting research into olfaction: How do you ‘materialize’ odors and by what criteria do you make them comparable? Part of the contemporary significance of research in olfaction is its potential for methodological innovations: How do you overcome the persistent difficulties in modeling transmembrane receptors and how should the organization of activation patterns in the cortex be modeled? By tracing the emergence, success, decline and failure of standard lab routines in past and present research on the sense of smell, I want to understand the uncertainties and the ‘missing knowledge’ that drive contemporary (neuro)scientific research.

Talk has three parts: quick general philosophy intro, odorant receptors and GPCRs, olfactory cortex mapping.
(Correction: Linda Buck tried 3 not 8 years finding the receptors.)

Retinal Pigment Epithelial cells (RPE) express both GPR143 and myocilin, which interact in a signal
transduction-dependent manner. In heterologous systems, activation of GPR143 with ligand causes
transient recruitment of myocilin to internalized receptors, which appears to be the entry point of
myocilin to the endocytic pathway. In some but not all cells, myocilin also traffics through the multivesicular
body (MVB) and is released on the surface of exosomes in a signal transduction-dependent
fashion. Little is known regarding the role of exosomes in RPE, but they likely serve as a mode of
communication between the RPE and the outer retina. In this study, we used posterior poles with retina
removed from fresh human donor eyes as a model to test the relationship between GPR143, myocilin,
and exosomes in an endogenous system. We isolated exosomes released by RPE using differential
centrifugation of media conditioned by the RPE for 25 min, and then characterized the exosomes using
nanoparticle tracking to determine the number and size of the exosomes. Next, we tested whether ligand
stimulation of GPR143 using L-DOPA altered RPE exosome release. Finally, we investigated whether
myocilin was present on the exosomes released by RPE and whether L-DOPA stimulation of GPR143
caused recruitment of myocilin to the endocytic pathway, as we have previously observed using cultured
cells. Activation of GPR143 halted RPE exosome release, while simultaneously recruiting myocilin to the
endocytic compartment. Together, our results indicate that GPR143 and myocilin function in a signal
transduction system that can control exosome release from RPE

Genomics and proteomics discovery is leading to the identification of all proteins and to the opportunity, and challenge, to reveal the protein recognition networks that drive virtually all biological processes. Over the past decade, biosensors have emerged as a key technology for detection and analysis of biomolecular interactions. An important limitation in developing such biosensors is that the focus has been mainly on sensor platforms, the transducing hardware that converts interaction signals into recorded data, without adequately considering the role of molecular interfaces, the elements of sensors that interact with analytes to produce signals. We have investigated this alternative focus by identifying and, where necessary, designing molecular interfaces that will more effectively drive new biosensor development and utilization in biomedical and biotechnological investigations. Here we describe our recent studies of coiled coil and lipid bilayer interfaces and the potential to use these to expand sensing technologies for multiplexed target detection and analysis in increasingly biologically relevant membrane like environments. Copyright © 2004 John Wiley & Sons, Ltd.

Neurobiology studies mechanisms of cell signalling. A key question is how cells recognise specific signals. In this context, olfaction has become an important experimental system over the past 25 years. The olfactory system responds to an array of structurally diverse stimuli. The discovery of the olfactory receptors (ORs), recognising these stimuli, established the olfactory pathway as part of a greater group of signalling mechanisms mediated by G-protein-coupled receptors (GPCRs). GPCRs are the largest protein family in the mammalian genome and involved in numerous fundamental physiological processes. The OR family exhibits two characteristics that make them an excellent model system to understand GPCRs: its size and the structural diversity of its members. Research on the OR binding site investigates what amino acid sequences determine the receptor binding capacity. This promises a better understanding of how the basic genetic makeup of GPCRs relates to their diversification in ligand binding capacities.

DNA fingerprinting by pulsed-field gel electrophoresis (PFGE) 1 is the methodology used by the PulseNet International network for worldwide surveillance of foodborne diseases [1]. Success relies on the participants' adherence to PulseNet standard working protocols that make ...

Many G protein-coupled receptor (GPCR) models have been built over the years. The release of the structure of bovine rhodopsin in August 2000 enabled us to analyze models built before that period to learn more about the models we build today. We conclude that the GPCR modelling field is riddled with ‘common knowledge’ similar to Lord Kelvin’s remark in 1895 that “heavier-than-air flying machines are impossible”, and we summarize what we think are the (im)possibilities of modelling GPCRs using the coordinates of bovine rhodopsin as a template. Associated WWW pages: http://www.gpcr.org/articles/2003_mod/.

The glutamatergic pathway has been consistently involved in the physiopathology of depressive disorder. However a complete dissection and integration of its role in the context of other known mechanisms is lacking. We summarized and integrated the evidence of various levels of interaction between glutamatergic and monoaminergic pathways (see videos). We identified six molecular pathways, some of which with specific regional

The human recombinant Interferon-alpha(2a) (IFNalpha(2a)) is a potent drug (Roferon-A) to treat various types of cancer and viral diseases including Hepatitis B/C infections. To improve the pharmacological properties of the drug, a new pegylated form of IFNalpha(2a) was developed (PEGASYS). This 40 kDa PEG-conjugated IFNalpha(2a) ((40)PEG-IFNalpha(2a)) is obtained by the covalent binding of one 40 kDa branched PEG-polymer to a lysine side chain of IFNalpha(2a). (40)PEG-IFNalpha(2a) is a mixture of mainly six monopegylated positional isomers modified at K31, K134, K131, K121, K164, and K70, respectively. Here we report the detailed structural and biophysical characterization of (40)PEG-IFNalpha(2a) and its positional isomers, in comparison with IFNalpha(2a), using NMR spectroscopy, analytical ultracentrifugation, circular dichroism, fluorescence spectroscopy, and differential scanning calorimetry. Our results show that the three-dimensional structure of IFNalpha(2a) is not modified b...