Systems analysis of RhoGEF and RhoGAP regulatory proteins reveals spatially organized RAC1 signalling from integrin adhesions.

Rho GTPases are central regulators of the cytoskeleton and, in humans, are controlled by 145 multidomain guanine nucleotide exchange factors (RhoGEFs) and GTPase-activating proteins (RhoGAPs). How Rho signalling patterns are established in dynamic cell spaces to control cellular morphogenesis is unclear. Through a family-wide characterization of substrate specificities, interactomes and localization, we reveal at the systems level how RhoGEFs and RhoGAPs contextualize and spatiotemporally control Rho signalling.

Native KCC2 interactome reveals PACSIN1 as a critical regulator of synaptic inhibition.

KCC2 is a neuron-specific K-Cl cotransporter essential for establishing the Cl gradient required for hyperpolarizing inhibition in the central nervous system (CNS). KCC2 is highly localized to excitatory synapses where it regulates spine morphogenesis and AMPA receptor confinement. Aberrant KCC2 function contributes to human neurological disorders including epilepsy and neuropathic pain.

Phosphoinositide 3-kinase enables phagocytosis of large particles by terminating actin assembly through Rac/Cdc42 GTPase-activating proteins.

Phagocytosis is responsible for the elimination of particles of widely disparate sizes, from large fungi or effete cells to small bacteria. Though superficially similar, the molecular mechanisms involved differ: engulfment of large targets requires phosphoinositide 3-kinase (PI3K), while that of small ones does not. Here, we report that inactivation of Rac and Cdc42 at phagocytic cups is essential to complete internalization of large particles.

A Rac/Cdc42 exchange factor complex promotes formation of lateral filopodia and blood vessel lumen morphogenesis.

During angiogenesis, Rho-GTPases influence endothelial cell migration and cell-cell adhesion; however it is not known whether they control formation of vessel lumens, which are essential for blood flow. Here, using an organotypic system that recapitulates distinct stages of VEGF-dependent angiogenesis, we show that lumen formation requires early cytoskeletal remodelling and lateral cell-cell contacts, mediated through the RAC1 guanine nucleotide exchange factor (GEF) DOCK4 (dedicator of cytokinesis 4). DOCK4 signalling is necessary for lateral filopodial protrusions and tubule remodelling prior to lumen formation, whereas proximal, tip filopodia persist in the absence of DOCK4.

Combinatorial proteomic analysis of intercellular signaling applied to the CD28 T-cell costimulatory receptor.

Systematic characterization of intercellular signaling approximating the physiological conditions of stimulation that involve direct cell-cell contact is challenging. We describe a proteomic strategy to analyze physiological signaling mediated by the T-cell costimulatory receptor CD28. We identified signaling pathways activated by CD28 during direct cell-cell contact by global analysis of protein phosphorylation.

Src homology 2 domain containing protein 5 (SH2D5) binds the breakpoint cluster region protein, BCR, and regulates levels of Rac1-GTP.

SH2D5 is a mammalian-specific, uncharacterized adaptor-like protein that contains an N-terminal phosphotyrosine-binding domain and a C-terminal Src homology 2 (SH2) domain. We show that SH2D5 is highly enriched in adult mouse brain, particularly in Purkinjie cells in the cerebellum and the cornu ammonis of the hippocampus. Despite harboring two potential phosphotyrosine (Tyr(P)) recognition domains, SH2D5 binds minimally to Tyr(P) ligands, consistent with the absence of a conserved Tyr(P)-binding arginine residue in the SH2 domain.

Protein interaction network of the mammalian Hippo pathway reveals mechanisms of kinase-phosphatase interactions.

The Hippo pathway regulates organ size and tissue homeostasis in response to multiple stimuli, including cell density and mechanotransduction. Pharmacological inhibition of phosphatases can also stimulate Hippo signaling in cell culture. We defined the Hippo protein-protein interaction network with and without inhibition of serine and threonine phosphatases by okadaic acid.

The CRAPome: a contaminant repository for affinity purification-mass spectrometry data.

Affinity purification coupled with mass spectrometry (AP-MS) is a widely used approach for the identification of protein-protein interactions. However, for any given protein of interest, determining which of the identified polypeptides represent bona fide interactors versus those that are background contaminants (for example, proteins that interact with the solid-phase support, affinity reagent or epitope tag) is a challenging task. The standard approach is to identify nonspecific interactions using one or more negative-control purifications, but many small-scale AP-MS studies do not capture a complete, accurate background protein set when available controls are limited.

Interaction domains of Sos1/Grb2 are finely tuned for cooperative control of embryonic stem cell fate.

Metazoan evolution involves increasing protein domain complexity, but how this relates to control of biological decisions remains uncertain. The Ras guanine nucleotide exchange factor (RasGEF) Sos1 and its adaptor Grb2 are multidomain proteins that couple fibroblast growth factor (FGF) signaling to activation of the Ras-Erk pathway during mammalian development and drive embryonic stem cells toward the primitive endoderm (PrE) lineage. We show that the ability of Sos1/Grb2 to appropriately regulate pluripotency and differentiation factors and to initiate PrE development requires collective binding of multiple Sos1/Grb2 domains to their protein and phospholipid ligands.

Salmonella exploits Arl8B-directed kinesin activity to promote endosome tubulation and cell-to-cell transfer.

The facultative intracellular pathogen Salmonella enterica serovar Typhimurium establishes a replicative niche, the Salmonella-containing vacuole (SCV), in host cells. Here we demonstrate that these bacteria exploit the function of Arl8B, an Arf family GTPase, during infection. Following infection, Arl8B localized to SCVs and to tubulated endosomes that extended along microtubules in the host cell cytoplasm.

The integral membrane of lysosomes: its proteins and their roles in disease.

The protein composition of the integral lysosomal membrane and the membrane-associated compartment have been defined in part by proteomics approaches. While the role of its constituent hydrolases in a large array of human disorders has been well-documented, the manner in which membrane proteins are integrated into the organelle, the multiprotein complexes that form at the organelle's cytosolic surface and their roles in the biogenesis and functional control of the organelle are now emerging. Defining cytosolic targeting complexes that affect the function of the lysosomal/endosomal compartment may help to identify the lysosome's role in a variety of human pathologies.

Cryptic splice site in the complementary DNA of glucocerebrosidase causes inefficient expression.

The low levels of human lysosomal glucocerebrosidase activity expressed in transiently transfected Chinese hamster ovary (CHO) cells were investigated. Reverse transcription PCR (RT-PCR) demonstrated that a significant portion of the transcribed RNA was misspliced owing to the presence of a cryptic splice site in the complementary DNA (cDNA). Missplicing results in the deletion of 179 bp of coding sequence and a premature stop codon.

Lysosomal membranes from beige mice contain higher than normal levels of endoplasmic reticulum proteins.

Chediak-Higashi syndrome is characterized by dysfunctional giant organelles of common origin, that is, lysosomes, melanosomes, and platelet dense bodies. Its defective gene LYST encodes a large molecular weight protein whose function is unknown. The Beige mouse also defective in Lyst is a good model of the human disease.

Identification of the gene encoding the enzyme deficient in mucopolysaccharidosis IIIC (Sanfilippo disease type C).

Mucopolysaccharidosis IIIC (MPS IIIC), or Sanfilippo C, represents the only MPS disorder in which the responsible gene has not been identified; however, the gene has been localized to the pericentromeric region of chromosome 8. In an ongoing proteomics study of mouse lysosomal membrane proteins, we identified an unknown protein whose human homolog, TMEM76, was encoded by a gene that maps to 8p11.1.

The Arf-family protein, Arl8b, is involved in the spatial distribution of lysosomes.

Lysosomes are late-endocytic organelles which primarily contribute to degradation and recycling of cellular material. From a previous proteomics study of purified rat liver lysosomal membranes we identified a protein from the Arf-family of small GTPases, Arl8b. Although proteins of the Arf-family have roles in a wide range of cellular functions, most notably roles in protein/vesicular trafficking, Arl8b represents the first from this protein family to be associated with a late-endocytic organelle.

Lysosomal membrane proteomics and biogenesis of lysosomes.

This review focuses on events involved in the biogenesis of the lysosome. This organelle contains a diverse array of soluble, luminal proteins capable of digesting all the macromolecules in the cell. Altered function of lysosomes or its constituent enzymes has been implicated in a host of human pathologies, including storage diseases, cancer, and infectious and neurodegenerative diseases.

Identification of the hydrophobic glycoproteins of Caenorhabditis elegans.

Hydrophobic proteins such as integral membrane proteins are difficult to separate, and therefore to study, at a proteomics level. However, the Asn-linked (N-linked) carbohydrates (N-glycans) contained in membrane glycoproteins are important in differentiation, embryogenesis, inflammation, cancer and metastasis, and other vital cellular processes. Thus, the identification of these proteins and their sites of glycosylation in a well-characterized model organism is the first step toward understanding the mechanisms by which N-glycans and their associated proteins function in vivo.

A proteomic analysis of lysosomal integral membrane proteins reveals the diverse composition of the organelle.

Lysosomes are endocytic subcellular compartments that contribute to the degradation and recycling of cellular material. Using highly purified rat liver tritosomes (Triton WR1339-filled lysosomes) and an ion exchange chromatography/LC-tandem MS-based protein/peptide separation and identification procedure, we characterized the major integral membrane protein complement of this organelle. While many of the 215 proteins we identified have been previously associated with lysosomes and endosomes, others have been associated with the endoplasmic reticulum, Golgi, cytosol, plasma membrane, and lipid rafts.

Membrane-associated estrogen receptor and caveolin-1 are present in central nervous system myelin and oligodendrocyte plasma membranes.

The estrogen receptor (ER) is a member of a superfamily of ligand-regulated transcription factors that were thought to localize primarily to the nucleus; however, a membrane-associated ER that can initiate rapid non-genomic cell-signaling events has been identified recently in various cells. The presence of the ER in myelin has not been reported although the nuclear form has been detected in oligodendrocytes. We have shown that an ER with similarities to ERbeta is present in isolated central nervous system (CNS) myelin, the myelin sheath in spinal cord and brain sections, and the oligodendrocyte plasma membrane using two-dimensional (2D) PAGE, mass spectrometry, peptide mass fingerprinting, Western blotting of 1D and 2D gels, and confocal microscopy.

Presenilin-1, nicastrin, amyloid precursor protein, and gamma-secretase activity are co-localized in the lysosomal membrane.

Alzheimer's disease (AD) is caused by the cerebral deposition of beta-amyloid (Abeta), a 38-43-amino acid peptide derived by proteolytic cleavage of the amyloid precursor protein (APP). Initial studies indicated that final cleavage of APP by the gamma-secretase (a complex containing presenilin and nicastrin) to produce Abeta occurred in the endosomal/lysosomal system. However, other studies showing a predominant endoplasmic reticulum localization of the gamma-secretase proteins and a neutral pH optimum of in vitro gamma-secretase assays have challenged this conclusion.

Nicastrin is a resident lysosomal membrane protein.

Nicastrin has been recently identified as part of the gamma-secretase complex that includes presenilin and other proteins. It is involved in the degradation of amyloid precursor protein to produce beta-amyloid peptides which are believed to be central to the pathophysiology of Alzheimer's disease. Previous reports have localized presenilin and nicastrin to the endoplasmic reticulum.

Novel mutations (Asn 484 Lys, Thr 500 Ala, Gly 438 Glu) in Morquio B disease.

Primary deficiency of beta-galactosidase results in GM1 gangliosidosis and Morquio B disease. Of the more than 40 disease-causing mutations described in the Gal gene to date, about 75% are of the missense type and are scattered along the length of the gene. No single, major common mutation has been associated with GM1 gangliosidosis.