Interaction proteomics of synapse protein complexes

The brain integrates complex types of information, and executes a wide range of physiological and behavioral processes. Trillions of tiny organelles, the synapses, are central to neuronal communication and information processing in the brain. Synaptic transmission involves an intricate network of synaptic proteins that forms the molecular machinery underlying transmitter release, activation, and modulation of transmitter receptors and signal transduction cascades. These processes are dynamically regulated and underlie neuroplasticity, crucial to learning and memory formation. In recent years, interaction proteomics has increasingly been used to elucidate the constituents of synaptic protein complexes. Unlike classic hypothesis-based assays, interaction proteomics detects both known and novel interactors without bias. In this trend article, we focus on the technical aspects of recent proteomics to identify synapse protein complexes, and the complementary methods used to verify the protein–protein interaction. Moreover, we discuss the experimental feasibility of performing global analysis of the synapse protein interactome

Proteomics, ultrastructure, and physiology of hippocampal synapses in a fragile X syndrome mouse model reveal presynaptic phenotype

Fragile X syndrome (FXS), the most common form of hereditary mental retardation, is caused by a loss-of-function mutation of the Fmr1 gene, which encodes fragile X mental retardation protein (FMRP). FMRP affects dendritic protein synthesis, thereby causing synaptic abnormalities. Here, we used a quantitative proteomics approach in an FXS mouse model to reveal changes in levels of hippocampal synapse proteins. Sixteen independent pools of Fmr1 knock-out mice and wild type mice were analyzed using two sets of 8-plex iTRAQ experiments. Of 205 proteins quantified with at least three distinct peptides in both iTRAQ series, the abundance of 23 proteins differed between Fmr1 knock-out and wild type synapses with a false discovery rate (q-value) <5%. Significant differences were confirmed by quantitative immunoblotting. A group of proteins that are known to be involved in cell differentiation and neurite outgrowth was regulated; they included Basp1 and Gap43, known PKC substrates, and Cend1. Basp1 and Gap43 are predominantly expressed in growth cones and presynaptic terminals. In line with this, ultrastructural analysis in developing hippocampal FXS synapses revealed smaller active zones with corresponding postsynaptic densities and smaller pools of clustered vesicles, indicative of immature presynaptic maturation. A second group of proteins involved in synaptic vesicle release was up-regulated in the FXS mouse model. In accordance, paired-pulse and short-term facilitation were significantly affected in these hippocampal synapses. Together, the altered regulation of presynaptically expressed proteins, immature synaptic ultrastructure, and compromised short-term plasticity points to presynaptic changes underlying glutamatergic transmission in FXS at this stage of development. © 2011 by The American Society for Biochemistry and Molecular Biology, Inc

An Sfi1-like centrin-interacting centriolar plaque protein affects nuclear microtubule homeostasis.

Malaria-causing parasites achieve rapid proliferation in human blood through multiple rounds of asynchronous nuclear division followed by daughter cell formation. Nuclear divisions critically depend on the centriolar plaque, which organizes intranuclear spindle microtubules. The centriolar plaque consists of an extranuclear compartment, which is connected via a nuclear pore-like structure to a chromatin-free intranuclear compartment. Composition and function of this non-canonical centrosome remain largely elusive. Centrins, which reside in the extranuclear part, are among the very few centrosomal proteins conserved in Plasmodium falciparum. Here we identify a novel centrin-interacting centriolar plaque protein. Conditional knock down of this Sfi1-like protein (PfSlp) caused a growth delay in blood stages, which correlated with a reduced number of daughter cells. Surprisingly, intranuclear tubulin abundance was significantly increased, which raises the hypothesis that the centriolar plaque might be implicated in regulating tubulin levels. Disruption of tubulin homeostasis caused excess microtubules and aberrant mitotic spindles. Time-lapse microscopy revealed that this prevented or delayed mitotic spindle extension but did not significantly interfere with DNA replication. Our study thereby identifies a novel extranuclear centriolar plaque factor and establishes a functional link to the intranuclear compartment of this divergent eukaryotic centrosome

Nbea interacts with SAP102 in HEK293T cells.

<p>Co-imunoprecipitation of Nbea and SAP102. (A) HEK 293T cells were co-transfected with full-length Nbea tagged with YFP and flag-tagged SAP102 or an empty vector and were immunoprecipitated (IP) with α-GFP antibody before immuno-blotting (IB) with α-Nbea and α-flag antibody. In the control condition non-coated, empty beads (EB) were used for the IP. (B) Reverse IP to the ones in A. HEK 293T cells were co-transfected with full-length Nbea tagged with YFP and flag-tagged SAP102 or an empty vector, but this time they were immuno-precipitated (IP) with α-flag antibody before immuno-blotting (IB) with α-Nbea and α-flag antibody. In the control condition non-coated, empty beads (EB) were used for the IP.</p

The E2218R mutation within the PH domain abolishes Nbea’s binding to SAP102.

<p>(A) HEK293 cells were co-transfected with flag-tagged SAP102 and either the non-mutated C-terminal part of Nbea (encompassing the Duf, PH, BEACH and WD40 domains) fused to GFP or the mutated versions of this protein. The following mutations were used: E2090K in the DUF domain (1), E2218R in the PH domain (2), N2302A in the BEACH domain (3), V2773I in the WD40 domain (4), and an additional three mutations within the BEACH domain, V2346Q (5), E2447R (6) and P2499S (7) (B) Quantification of FLAG-tagged and GFP-tagged protein levels of the immuno-blot. Error bars indicate the standard error of the mean (SEM).</p

List of proteins identified from IPs on P2+ microsomes fraction from P84 mice.

<p>The experiments were performed on P2+ microsomes fraction from P84 WT mice using n-Dodecyl β-D-maltoside (DDM) as detergent for protein extraction. Proteins listed in this table were present in at least 2 of the 3 IP experiments, and were enriched at least 20-fold in the IP samples compared to the controls. The proteins in bold were also identified in IPs performed on brain lysates of E18 Nbea mice. The “unused” value is a summation of protein scores from all non-redundant peptides matched to a single protein. Proteins with “unused” value <1.3 have low confidence and were excluded from the analysis.</p

SAP102 binds to the C-terminal part of Nbea.

<p>(A) HEK293 cells were co-transfected with flag-tagged SAP102 and either full-length Nbea tagged with YFP or various Nbea deletions encompassing different domains fused to GFP. Following deletions were used: GFP-Duf, PH, BEACH, WD40; GFP-Duf, PH, BEACH; GFP-PH, BEACH, WD40; GFP-PH, BEACH; GFP-PH; GFP-BEACH. In the control condition SAP-102 was co-transfected with YFP and GFP. IPs were performed using the α-GFP antibody, before immune-blotting with α-flag and α-GFP antibodies. (B) Quantification of flag and GFP protein levels using the immuno-labelled bands. Error bars indicate the standard error of the mean (SEM).</p

Previously identified binding partners of Nbea.

<p>Previously identified binding partners of Nbea.</p

Nbea interacts with a fraction of SAP102 in vivo.

<p>(A) Schematic representation of mouse Nbea (NCBI Reference Sequence: NP_085098.1). The predicted armadillo (ARM) repeat-flanked Concavanalin A (Con A)-like lectin domain is localized at the N-terminus of the protein (blue). The region the Nbea antibody was raised against is depicted by the purple rectangle, encompassing also the PKA binding site (pink stripe). At the C-terminus the domain of unknown function 1088 (DUF; in orange), the Pleckstrin-Homology like domain (PH; in gray), the BEACH domain (yellow) and the WD40 repeats (red) are depicted. (B) Co-immunoprecipitation of SAP102 and Nbea from crude membrane with microsomes fraction (P2+M) of P84 WT mice. Proteins were immunoprecipitated (IP) with two different α-SAP102 antibodies, i.e. a mouse monoclonal (NeuroMab clone N19/2; left lane) and a rabbit polyclonal one (GenScript; right lane), respectively. In the control condition non-coated, empty beads (EB) were used for the IP. The <i>Input</i> lane represents the crude membrane with microsomes fraction that was used for immunoprecipitation. Immuno-blotting (IB) was performed with α-Nbea and α-SAP102 antibody (NeuroMab clone N19/2). (C) Dendritic Nbea immunoreactivity shows limited overlap with SAP102. DIV14 WT hippocampal neurons (E18) fixed in methanol and stained for endogenous SAP102 (in green), endogenous Nbea (in red) and MAP2 (not shown in the merge). Top scale bar  = 20 µm, lower scale bar  = 5 µm.</p