Reconstructing protein complexes: From proteomics to systems biology

Armstrong, J. Douglas; Pocklington, Andrew; Cumiskey, Mark; Grant, Seth G. N.

doi:10.1002/pmic.200500895

Cited by 19 publications

(10 citation statements)

References 25 publications

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“…Moreover, because interactions between proteins depend on conditions defined by the experimental paradigm, such as those imposing constraints on the stability of protein bonds as well as the proximity of interaction motifs (36), it is expected that NIPs identified by pulldown will differ from NIPs identified by immunoprecipitation (33,37). Clearly, the combined use of multiple techniques provides the best strategy for identifying the full spectrum of interactions for any given protein (38). This fact is underscored by a report on the proteomic analysis of the NMDA receptor from brain, showing Ͼ77 binding partners for this receptor by use of immunoprecipitation, pulldown, and yeast two-hybrid procedures (19).…”

Section: Discussionmentioning

confidence: 99%

Intracellular complexes of the β2 subunit of the nicotinic acetylcholine receptor in brain identified by proteomics

Kabbani

Woll

Levenson

et al. 2007

Proc. Natl. Acad. Sci. U.S.A.

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Nicotine acetylcholine receptors (nAChRs) comprise a family of ligand-gated channels widely expressed in the mammalian brain. The ␤2 subunit is an abundant protein subunit critically involved in the cognitive and behavioral properties of nicotine as well as in the mechanisms of nicotine addiction. In this work, we used matrixassisted laser desorption ionization time-of-flight tandem mass spectrometry (MALDI-TOF-TOF MS/MS) to uncover protein interactions of the intracellular loop of the ␤2 subunit and components of immunoprecipitated ␤2-nAChR complexes from mouse brain. Using the ␤2-knockout mouse to exclude nonspecific binding to the ␤2 antibody, we identify 21 nAChR-interacting proteins (NIPs) expressed in brain. Western blot analysis confirmed the association between the ␤2 subunit and candidate NIPs. Based on their functional profiles, the hypothesis is suggested that the identified NIPs can regulate the trafficking and signaling of the ␤2-nAChR. Interactions of the ␤2 subunit with NIPs such as G protein ␣, G protein-regulated inducer of neurite outgrowth 1, and G proteinactivated K ؉ channel 1 suggest a link between nAChRs and cellular G protein pathways. These findings reveal intracellular interactions of the ␤2 subunit and may contribute to the understanding of the mechanisms of nAChR signaling and trafficking in neurons.mass spectrometry ͉ receptor complex N icotine is a common drug of addiction and a leading cause of preventable deaths in developed countries (1, 2). The target of nicotine is a class of nicotinic acetylcholine receptors (nAChRs) existing as pentameric channels made up of various receptor subunits and distributed throughout the brain (3). To date, 11 neuronal subunits have been identified: ␣2-␣9 and ␤2-␤4. The specific combination of these subunits confers the pharmacological and physiological properties of the nAChR (4). Studies show that Ϸ90% of the high-affinity nicotine receptor sites within the brain consist of the ␣4␤2-nAChR (5, 6). Topological analysis of nAChR subunits indicates the presence of a large, highly divergent intracellular loop (M3-M4 loop) that is implicated in trafficking and targeting properties of nAChRs (7,8). Recent analysis of a prokaryotic homolog of the nAChR family reveals that the M3-M4 loop is unique to the evolution of the nAChR in eukaryotes (9).The generation of subunit-specific knockout (KO) mice has proven valuable in defining the role of individual nAChR subunits in brain physiology and animal behavior (10). Experiments in mice lacking the ␤2 subunit (␤2 Ϫ/Ϫ ) demonstrate an important role for this receptor subunit in neuronal development and plasticity (10), protection from excitotoxicity (11), and the mechanisms underlying cognitive and social behaviors (12). ␤2 Ϫ/Ϫ mice also exhibit a loss of nicotine self-administration and nicotine-elicited firing and dopamine release from dopaminergic neurons of the ventral tegmental area (13,14). Because reexpression of the ␤2 subunit in ␤2 Ϫ/Ϫ mice was found sufficient to restore the electrophysiological and beh...

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Section: Discussionmentioning

confidence: 99%

Intracellular complexes of the β2 subunit of the nicotinic acetylcholine receptor in brain identified by proteomics

Kabbani

Woll

Levenson

et al. 2007

Proc. Natl. Acad. Sci. U.S.A.

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“…In order to synthesize the data from proteomic studies in a meaningful way, a range of computational techniques can be employed [26]. Information from the biochemical literature, particularly protein-protein interaction (PPI) data, can be applied to increase our understanding of functional pathways within a cellular compartment of interest.…”

Section: Introductionmentioning

confidence: 99%

Systems approach to explore components and interactions in the presynapse

et al. 2009

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The application of proteomic techniques to neuroscientific research provides an opportunity for a greater understanding of nervous system structure and function. As increasing amounts of neuroproteomic data become available, it is necessary to formulate methods to integrate these data in a meaningful way to obtain a more comprehensive picture of neuronal subcompartments. Furthermore, computational methods can be used to make biologically relevant predictions from large proteomic datasets. Here, we applied an integrated proteomics and systems biology approach to characterize the presynaptic nerve terminal. For this, we carried out proteomic analyses of presynaptically enriched fractions, and generated a presynaptic literature-based protein-protein interaction (PPI) network. We combined these with other proteomic analyses to generate a core list of 117 presynaptic proteins, and used graph theory-inspired algorithms to predict 92 additional components and a presynaptic complex containing 17 proteins. Some of these predictions were validated experimentally, indicating that the computational analyses can identify novel proteins and complexes in a subcellular compartment. We conclude that the combination of techniques (proteomics, data integration, and computational analyses) used in this study are useful in obtaining a comprehensive understanding of functional components, especially low-abundance entities and/or interactions in the presynaptic nerve terminal.

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“…On a larger scale, existing proteomic methods, such as mass spectrometry and protein array tools, can be applied to reveal protein–protein interactions (Husi et al, 2000; Schweitzer et al, 2003; Yuk et al, 2004; Collins and Choudhary, 2008). Bioinformatics tools could also be employed to generate hypothesis of interactions that could be verified experimentally and to construct interaction maps and models that could be used to predict the effects of mutations of single proteins (Armstrong et al, 2006). …”

Section: A Molecular Catalog Of Inhibitory Synapsesmentioning

confidence: 99%

Understanding the Molecular Diversity of GABAergic Synapses

Sassoè‐Pognetto¹,

Frola²,

Pregno³

et al. 2011

Front. Cell. Neurosci.

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GABAergic synapses exhibit a high degree of subcellular and molecular specialization, which contrasts with their apparent simplicity in ultrastructural appearance. Indeed, when observed in the electron microscope, GABAergic synapses fit in the symmetric, or Gray’s type II category, being characterized by a relatively simple postsynaptic specialization. The inhibitory postsynaptic density cannot be readily isolated, and progress in understanding its molecular composition has lagged behind that of excitatory synapses. However, recent studies have brought significant progress in the identification of new synaptic proteins, revealing an unexpected complexity in the molecular machinery that regulates GABAergic synaptogenesis. In this article, we provide an overview of the molecular diversity of GABAergic synapses, and we consider how synapse specificity may be encoded by selective trans-synaptic interactions between pre- and postsynaptic adhesion molecules and secreted factors that reside in the synaptic cleft. We also discuss the importance of developing cataloguing tools that could be used to decipher the molecular diversity of synapses and to predict alterations of inhibitory transmission in the course of neurological diseases.

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Reconstructing protein complexes: From proteomics to systems biology

Cited by 19 publications

References 25 publications

Intracellular complexes of the β2 subunit of the nicotinic acetylcholine receptor in brain identified by proteomics

Intracellular complexes of the β2 subunit of the nicotinic acetylcholine receptor in brain identified by proteomics

Systems approach to explore components and interactions in the presynapse

Understanding the Molecular Diversity of GABAergic Synapses

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