We performed a systematic, large-scale analysis of human protein complexes comprising gene products implicated in many different categories of human disease to create a phenome-interactome network. This was done by integrating quality-controlled interactions of human proteins with a validated, computationally derived phenotype similarity score, permitting identification of previously unknown complexes likely to be associated with disease. Using a phenomic ranking of protein complexes linked to human disease, we developed a Bayesian predictor that in 298 of 669 linkage intervals correctly ranks the known disease-causing protein as the top candidate, and in 870 intervals with no identified disease-causing gene, provides novel candidates implicated in disorders such as retinitis pigmentosa, epithelial ovarian cancer, inflammatory bowel disease, amyotrophic lateral sclerosis, Alzheimer disease, type 2 diabetes and coronary heart disease. Our publicly available draft of protein complexes associated with pathology comprises 506 complexes, which reveal functional relationships between disease-promoting genes that will inform future experimentation.

The neural cell adhesion molecule (NCAM) promotes axonal outgrowth, presumably through an interaction with the fibroblast growth factor receptor (FGFR). NCAM also has a little-understood ATPase activity. We here demonstrate for the first time a direct interaction between NCAM (fibronectin type III [F3] modules 1 and 2) and FGFR1 (Ig modules 2 and 3) by surface plasmon resonance (SPR) analysis. The structure of the NCAM F3 module 2 was determined by NMR and the module was shown by NMR to interact with the FGFR1 Ig module 3 and ATP. The NCAM sites binding to FGFR and ATP were found to overlap and ATP was shown by SPR to inhibit the NCAM-FGFR binding, indicating that ATP probably regulates the NCAM-FGFR interaction. Furthermore, we demonstrate that the NCAM module was able to induce activation (phosphorylation) of FGFR and to stimulate neurite outgrowth. In contrast, ATP inhibited neurite outgrowth induced by the module.

Lysosomal storage diseases (LSDs) often manifest with severe systemic and central nervous system (CNS) symptoms. The existing treatment options are limited and have no or only modest efficacy against neurological manifestations of disease. We demonstrate that recombinant human heat shock protein 70 (HSP70) improves the binding of several sphingolipid-degrading enzymes to their essential cofactor bis(monoacyl)glycerophosphate in vitro. HSP70 treatment reversed lysosomal pathology in primary fibroblasts from 14 patients with eight different LSDs. HSP70 penetrated effectively into murine tissues including the CNS and inhibited glycosphingolipid accumulation in murine models of Fabry disease (Gla(-/-)), Sandhoff disease (Hexb(-/-)), and Niemann-Pick disease type C (Npc1(-/-)) and attenuated a wide spectrum of disease-associated neurological symptoms in Hexb(-/-) and Npc1(-/-) mice. Oral administration of arimoclomol, a small-molecule coinducer of HSPs that is currently in clinical trials for Niemann-Pick disease type C (NPC), recapitulated the effects of recombinant human HSP70, suggesting that heat shock protein-based therapies merit clinical evaluation for treating LSDs.

Signal transduction by receptor tyrosine kinases is initiated by recruitment of a variety of signaling proteins to tyrosine-phosphorylated motifs in the activated receptors. Several signaling pathways are thus activated in parallel, the combination of which decides the cellular response. Here, we present a dual strategy for extensive mapping of tyrosine-phosphorylated proteins and probing of signal-dependent protein interactions of a signaling cascade. The approach relies on labeling of cells with "heavy" and "light" isotopic forms of Arg to distinguish two cell populations. First, tyrosine-phosphorylated proteins from stimulated ("heavy"-labeled) and control samples ("normal"-labeled) are isolated and subjected to high sensitivity Fourier transform ion cyclotron resonance mass spectrometry analysis. Next, phosphopeptides corresponding to tyrosine phosphorylation sites identified during the tyrosine phosphoproteomic analysis are used as baits to isolate phosphospecific protein binding partners, which are subsequently identified by mass spectrometry. We used this approach to identify 28 components of the signaling cascade induced by stimulation with the basic fibroblast growth factor. Insulin receptor substrate-4 was identified as a novel candidate in fibroblast growth factor receptor signaling, and we defined phosphorylation-dependent interactions with other components, such as adaptor protein Grb2, of the signaling cascade. Finally, we present evidence for a complex containing insulin receptor substrate-4 and ShcA in signaling by the fibroblast growth factor receptor. Fibroblast growth factors (FGFs)1 stimulate differentiation, proliferation, and other cellular events by inducing activation of the intrinsic tyrosine kinase in membrane-spanning receptors of the FGF receptor (FGFR) family. As receptors become activated, they autophosphorylate on several residues outside the kinase domain, thus allowing recruitment of cytosolic substrates and initiation of multiple downstream signaling pathways in parallel. The FGFR has been shown to initiate signaling through direct binding of substrates such as phospholipase C␥ (PLC␥) (1, 2), FGFR substrate-2 (FRS2) (3, 4), and ShcA (5). The end point of FGFR activation is defined in a cell-specific manner by the integration of these signaling pathways. A thorough understanding of cellular signaling processes requires the identification of the components of the entire signaling network followed by studies of the functional role of individual proteins. Large scale analysis of post-translationally modified (e.g. phosphorylated) proteins by mass spectrometry (MS) has gained momentum with the development of high resolution and high sensitivity hybrid mass spectrometers (e.g. quadrupole time-offlight (qTOF) instruments) and now constitutes a powerful functional proteomics platform for the analysis of protein networks of signaling processes (6).Since signaling proteins are not all "turned off" in the quiescent cell, and the stoichiometry of activation may vary according to the stimulu...

Neural cell adhesion molecule (NCAM) was originally characterised as a homophilic cell adhesion molecule (CAM) abundantly expressed in the nervous system. However, the last decade of research has challenged the traditional view and defined novel roles for NCAM. NCAM is now considered a signaling receptor that responds to both homophilic and heterophilic cues, as well as a mediator of cell-cell adhesion. This review describes NCAM function at the molecular level. We discuss recent models for extracellular ligand-interactions of NCAM, and the intracellular signaling cascade that follows to define cellular outcomes such as neurite outgrowth.

Overexpression of the fibroblast growth factor receptor-1 (FGFR-1), a prototypic receptor tyrosine kinase, is a feature of several human tumors. In human 293 cells overexpression of the FGFR-1 leads to constitutive activation of the receptor with concomitant sustained high increase in the cellular level of phosphotyrosine-containing proteins. Here we use mass spectrometry to study the tyrosinephosphorylated proteins induced by overexpression of the FGFR-1. Several well known components of FGFR-1 signaling were identified along with two novel candidates: NS-1-associated protein-1 and target of Myb 1-like protein. We subsequently applied mass spectrometry precursor ion scanning to identify 22 tyrosine phosphorylation sites distributed on six substrate proteins of the FGFR-1 or downstream tyrosine kinases. Novel in vivo tyrosine phosphorylation sites were found in the FGFR-1, phospholipase C␥, p90 ribosomal S6 kinase, cortactin, and NS-1-associated protein-1 as a result of sustained FGFR-1 signaling, and we propose these as functional links to downstream molecular and cellular processes. Receptor tyrosine kinases convey extracellular stimuli to intracellular signaling. When activated they induce tyrosine phosphorylation of specific cytosolic substrates and thereby create highly specific binding sites for the Src homology 2 (SH2) 1 or phosphotyrosine (Tyr(P)) binding domains in proteins that will further promote intracellular signaling cascades (1). The human genome encodes 58 different receptor tyrosine kinases (2), which are involved in events such as proliferation, differentiation, metabolic control, and migration.Overexpression of receptor tyrosine kinases can lead to constitutive activation as first described for the ErbB2 receptor (3) and to concomitant elevated cellular tyrosine phosphorylation status. Overexpression and amplification of the fibroblast growth factor receptor-1 (FGFR-1), a prototypic receptor tyrosine kinase, have been found in tumor samples from human pancreas (4, 5), breast (6, 7), brain (8, 9), prostate (10), thyroid gland (11), and salivary gland (12). To understand the role of FGFR-1 in cancer it is vital to define the signaling pathways that confer the oncogenic potential and how they differ from the "healthy" situation.In a recent study, immunoprecipitation with anti-Tyr(P) antibodies combined with SDS-PAGE and mass spectrometry was used to identify proteins that were tyrosine-phosphorylated as a result of epidermal growth factor stimulation of HeLa cells (13). Two novel candidates in the extensively studied epidermal growth factor receptor signaling pathway were isolated, and substrates present in femtomole amounts were identified. Techniques for tracing phosphopeptides in complex mixtures by MS have been developed and are now finding applications in phosphorylation studies of in vivo samples (14). These developments, however, have primarily been used in the characterization of serine and threonine phosphorylations due to (i) their relative abundance (Ͼ99.9%) compared with tyrosine pho...

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Recent proteomic efforts have created an extensive inventory of the human nucleolar proteome. However, approximately 30% of the identified proteins lack functional annotation. We present an approach of assigning function to uncharacterized nucleolar proteins by data integration coupled to a machine-learning method. By assembling protein complexes, we present a first draft of the human ribosome biogenesis pathway encompassing 74 proteins and hereby assign function to 49 previously uncharacterized proteins. Moreover, the functional diversity of the nucleolus is underlined by the identification of a number of protein complexes with functions beyond ribosome biogenesis. Finally, we were able to obtain experimental evidence of nucleolar localization of 11 proteins, which were predicted by our platform to be associates of nucleolar complexes. We believe other biological organelles or systems could be "wired" in a similar fashion, integrating different types of data with high-throughput proteomics, followed by a detailed biological analysis and experimental validation.