Mogens M. Nielsen

Brain Glutamate Transporter Proteins Form Homomultimers

Removal of excitatory amino acids from the extracellular fluid is essential for synaptic transmission and for avoiding excitotoxicity. The removal is accomplished by glutamate transporters located in the plasma membranes of both neurons and astroglia. The uptake system consists of several different transporter proteins that are carefully regulated, indicating more refined functions than simple transmitter inactivation. Here we show by chemical cross-linking, followed by electrophoresis and immunoblotting, that three rat brain glutamate transporter proteins (GLAST, GLT and EAAC) form homomultimers. The multimers exist not only in intact brain membranes but also after solubilization and after reconstitution in liposomes. Increasing the cross-linker concentration increased the immunoreactivity of the bands corresponding to trimers at the expense of the dimer and monomer bands. However, the immunoreactivities of the dimer bands did not disappear, indicating a mixture of dimers and trimers. GLT and GLAST do not complex with each other, but as demonstrated by double labeling post-embedding electron microscopic immunocytochemistry, they co-exist side by side in the same astrocytic cell membranes. The oligomers are held together noncovalently in vivo. In vitro, oxidation induces formation of covalent bonds (presumably -S-S-) between the subunits of the oligomers leading to the appearance of oligomer bands on SDS-polyacrylamide gel electrophoresis. Immunoprecipitation experiments suggest that GLT is the quantitatively dominant glutamate transporter in the brain. Radiation inactivation analysis gives a molecular target size of the functional complex corresponding to oligomeric structure. We postulate that the glutamate transporters operate as homomultimeric complexes.

Identification of Autophagosome-associated Proteins and Regulators by Quantitative Proteomic Analysis and Genetic Screens

Autophagy is one of the major intracellular catabolic pathways, but little is known about the composition of autophagosomes. To study the associated proteins, we isolated autophagosomes from human breast cancer cells using two different biochemical methods and three stimulus types: amino acid deprivation or rapamycin or concanamycin A treatment. The autophagosome-associated proteins were dependent on stimulus, but a core set of proteins was stimulus-independent. Remarkably, proteasomal proteins were abundant among the stimulus-independent common autophagosome-associated proteins, and the activation of autophagy significantly decreased the cellular proteasome level and activity supporting interplay between the two degradation pathways. A screen of yeast strains defective in the orthologs of the human genes encoding for a common set of autophagosome-associated proteins revealed several regulators of autophagy, including subunits of the retromer complex. The combined spatiotemporal proteomic and genetic data sets presented here provide a basis for further characterization of autophagosome biogenesis and cargo selection.

Inhibition of Adipocyte Differentiation by Resistin-like Molecule α BIOCHEMICAL CHARACTERIZATION OF ITS OLIGOMERIC NATURE

A novel family of cysteine-rich secreted proteins with unique tissue distribution has recently been identified. One of the members, resistin (for “resistance to insulin”), also called FIZZ3, was identified in a screen for molecules that are down-regulated in mature adipocytes upon administration of thiazolidinediones. The prototypical member of this family was originally identified from bronchoalveolar lavage fluid of inflamed lungs and designated FIZZ1 (“found in inflammatory zone”). This molecule was also found to be highly expressed in adipose tissue and was named resistin-like molecule α (RELMα). Here we demonstrate that RELMα inhibits the differentiation of 3T3-L1 preadipocytes into adipocytes. RELMα has no effect on proliferation of 3T3-L1 preadipocytes. Pretreatment of 3T3-L1 preadipocytes with RELMα does not affect insulin- or platelet-derived growth factor-induced mitogenesis. IRS-1 phosphorylation and glucose transport stimulated by insulin in mature adipocytes were also unaffected by RELMα. We show that RELMα forms disulfide-linked homooligomers based on results from electrophoresis under reducing and nonreducing conditions, coimmunoprecipitation experiments as well as by mass spectrometry. In addition, RELMα is able to form heterooligomers with resistin but not RELMβ. Since RELMα is expressed by adipose tissue and it is a secreted factor, our findings suggest that RELMα may be involved in the control of the adipogenesis as well as in the process of muscle differentiation.

StUbEx: Stable tagged ubiquitin exchange system for the global investigation of cellular ubiquitination.

Post-translational modification of proteins with the small polypeptide ubiquitin plays a pivotal role in many cellular processes, altering protein lifespan, location, and function and regulating protein-protein interactions. Ubiquitination exerts its diverse functions through complex mechanisms by formation of different polymeric chains and subsequent recognition of the ubiquitin signal by specific protein interaction domains. Despite some recent advances in the analytical tools for the analysis of ubiquitination by mass spectrometry, there is still a need for additional strategies suitable for investigation of cellular ubiquitination at the proteome level. Here, we present a stable tagged ubiquitin exchange (StUbEx) cellular system in which endogenous ubiquitin is replaced with an epitope-tagged version, thereby allowing specific and efficient affinity purification of ubiquitinated proteins for global analyses of protein ubiquitination. Importantly, the overall level of ubiquitin in the cell remains virtually unchanged, thus avoiding ubiquitination artifacts associated with overexpression. The efficiency and reproducibility of the method were assessed through unbiased analysis of epidermal growth factor (EGF) signaling by quantitative mass spectrometry, covering over 3400 potential ubiquitinated proteins. The StUbEx system is applicable to virtually any cell line and can be readily adapted to any of the ubiquitin-like post-translational modifications.

Cloning of rat thymic stromal lymphopoietin receptor (TSLPR) and characterization of genomic structure of murine Tslpr gene

Thymic stromal derived lymphopoietin receptor (TSLPR) is a novel receptor subunit that is related in sequence to the interleukin (IL)-2 receptor common gamma chain. TSLPR forms a heterodimeric complex with the IL-7 receptor alpha chain to form the receptor for thymic stromal derived lymphopoietin, a cytokine involved in B- and T-cell function. We have cloned the TSLP receptor from rat and find that the WSXWX motif commonly found in extracellular domains of cytokine receptors is conserved as a W(T/S)XV(T/A) motif among TSLP receptors from mouse, rat and human. As in the mouse, TSLP receptor is widely expressed in rats suggesting that TSLPR may have roles in signaling outside the hematopoietic system. A zooblot analysis revealed that TSLPR is expressed in all vertebrate species examined. The absence of TSLPR in Saccharomyces cerevisiae, Drosophila melanogaster and Caenorhabditis elegans genomes is similar to the expression of several other cytokine receptors that have been characterized thus far. We have also characterized the genomic structure of the murine Tslpr gene which shows that in addition to primary sequence homology, it shares a common genomic organization of coding exons with the murine IL-2 receptor common gamma chain (Il2rg). Use of an alternative splice acceptor site leads to two alternatively spliced transcript variants of murine TSLPR, both of which are functional receptors. Finally, using linkage analysis, we mapped the murine Tslpr gene to mouse chromosome 5 between the Ecm2 and Pxn genes.

UbiSite approach for comprehensive mapping of lysine and N-terminal ubiquitination sites

Ubiquitination is a post-translational modification (PTM) that is essential for balancing numerous physiological processes. To enable delineation of protein ubiquitination at a site-specific level, we generated an antibody, denoted UbiSite, recognizing the C-terminal 13 amino acids of ubiquitin, which remain attached to modified peptides after proteolytic digestion with the endoproteinase LysC. Notably, UbiSite is specific to ubiquitin. Furthermore, besides ubiquitination on lysine residues, protein N-terminal ubiquitination is readily detected as well. By combining UbiSite enrichment with sequential LysC and trypsin digestion and high-accuracy MS, we identified over 63,000 unique ubiquitination sites on 9,200 proteins in two human cell lines. In addition to uncovering widespread involvement of this PTM in all cellular aspects, the analyses reveal an inverse association between protein N-terminal ubiquitination and acetylation, as well as a complete lack of correlation between changes in protein abundance and alterations in ubiquitination sites upon proteasome inhibition.Using UbiSite, an antibody-based approach that specifically detects protein lysine and N-terminal ubiquitination, Blagoev and colleagues uncover lack of correlation between changes in protein ubiquitination and abundance upon proteasome inhibition.