Several hundred proteins become insoluble and aggregation-prone as a consequence of aging in Caenorhabditis elegans. The data indicate that these proteins influence disease-related protein aggregation and toxicity.

SUMMARY The nearly 600 proteases in the human genome regulate a diversity of biological processes, including programmed cell death. Comprehensive characterization of protease signaling in complex biological samples is limited by available proteomic methods. We have developed a general approach for global identification of proteolytic cleavage sites based on enzymatic biotinylation of free protein N-termini and positive enrichment of corresponding N-terminal peptides. Using this method to study apoptosis, we have sequenced 333 caspase-like cleavage sites distributed among 292 protein substrates. These sites are generally not predicted by in vitro caspase substrate specificity, but can be used to predict other physiological caspase cleavage sites. Structural bioinformatic studies show that caspase cleavage sites often appear in surface accessible loops and even occasionally in helical regions. Strikingly, we also find that a disproportionate number of caspase substrates physically interact, suggesting that these dimeric proteases target protein complexes and networks to elicit apoptosis.

O-linked N-acetylglucosamine (O-GlcNAc) is a dynamic, reversible monosaccharide modifier of serine and threonine residues on intracellular protein domains. Crosstalk between O-GlcNAcylation and phosphorylation has been hypothesized. Here, we identified over 1750 and 16,500 sites of O-GlcNAcylation and phosphorylation from murine synaptosomes, respectively. In total, 135 (7%) of all O-GlcNAcylation sites were also found to be sites of phosphorylation. Although many proteins were extensively phosphorylated and minimally O-GlcNAcylated, proteins found to be extensively O-GlcNAcylated were almost always phosphorylated to a similar or greater extent, indicating the O-GlcNAcylation system is specifically targeting a subset of the proteome that is also phosphorylated. Both PTMs usually occur on disordered regions of protein structure, within which, the location of O-GlcNAcylation and phosphorylation is virtually random with respect to each other, suggesting that negative crosstalk at the structural level is not a common phenomenon. As a class, protein kinases are found to be more extensively O-GlcNAcylated than proteins in general, indicating the potential for crosstalk of phosphorylation with O

SUMMARY Glutamate receptors play major roles in excitatory transmission in the vertebrate brain. Among ionotropic glutamate receptors (AMPA, kainate, NMDA), AMPA receptors mediate fast synaptic transmission and require TARP auxiliary subunits. NMDA receptors and kainate receptors play roles in synaptic transmission, but it remains uncertain whether these ionotropic glutamate receptors also have essential subunits. Using a proteomic screen, we have identified NETO2, a brain-specific protein of unknown function, as an interactor with kainate-type glutamate receptors. NETO2 modulates the channel properties of recombinant and native kainate receptors without affecting trafficking of the receptors and also modulates kainate-receptor-mediated mEPSCs. Furthermore, we found that kainate receptors regulate the surface expression of NETO2 and that NETO2 protein levels and surface expression are decreased in mice lacking the kainate receptor GluR6. The results show that NETO2 is a kainate receptor subunit with significant effects on glutamate signaling mechanisms in brain.

O-GlcNAc is a widespread dynamic carbohydrate modification of cytosolic and nuclear proteins with features analogous to phosphorylation. O-GlcNAc acts critically in many cellular processes, including signal transduction, protein degradation, and regulation of gene expression. However, the study of its specific regulatory functions has been limited by difficulties in mapping sites of O-GlcNAc modification. We report methods for direct enrichment and identification of in vivo O-GlcNAc-modified peptides through lectin weak affinity chromatography (

The microtubule-associated protein tau has been implicated in the pathogenesis of Alzheimer's disease (AD) and other neurodegenerative disorders. Reducing tau levels ameliorates AD-related synaptic, network, and behavioral abnormalities in transgenic mice expressing human amyloid precursor protein (hAPP). We used mass spectrometry to characterize the post-translational modification of endogenous tau isolated from wild-type and hAPP mice. We identified seven types of tau modifications at 63 sites in wild-type mice. Wild-type and hAPP mice had similar modifications, supporting the hypothesis that neuronal dysfunction in hAPP mice is enabled by physiological forms of tau. Our findings provide clear evidence for acetylation and ubiquitination of the same lysine residues; some sites were also targeted by lysine methylation. Our findings refute the hypothesis of extensive O-linked N-acetylglucosamine (O-GlcNAc) modification of endogenous tau. The complex post-translational modification of physiological tau suggests that tau is regulated by diverse mechanisms.

In the mammalian central nervous system, the structure known as the postsynaptic density (PSD) is a dense complex of proteins whose function is to detect and respond to neurotransmitter released from presynaptic axon terminals. Regulation of protein phosphorylation in this molecular machinery is critical to the activity of its components, which include neurotransmitter receptors, kinases/ phosphatases, scaffolding molecules, and proteins regulating cytoskeletal structure. To characterize the phosphorylation state of proteins in PSD samples, we combined strong cation exchange (SCX) chromatography with IMAC. Initially, tryptic peptides were separated by cation exchange and analyzed by reverse phase chromatography coupled to tandem mass spectrometry, which led to the identification of phosphopeptides in most SCX fractions. Because each of these individual fractions was too complex to characterize completely in single LC-MS/MS runs, we enriched for phosphopeptides by performing IMAC on each SCX fraction, yielding at least a 3-fold increase in identified phosphopeptides relative to either approach alone (SCX or IMAC). This enabled us to identify at least one site of phosphorylation on 23% (287 of 1,264) of all proteins found to be present in the postsynaptic density preparation. In total, we identified 998 unique phosphorylated peptides, mapping to 723 unique sites of phosphorylation. At least one exact site of phosphorylation was determined on 62% (621 of 998) of all phosphopeptides, and ϳ80% of identified phosphorylation sites are novel. Molecular & Cellular Proteomics 5: 914 -922, 2006.

SUMMARY Cells allocate substantial resources towards monitoring levels of nutrients that can be used for ATP generation by mitochondria. Among the many specialized cell types, neurons are particularly dependent on mitochondria due to their complex morphology and regional energy needs. Here, we report a molecular mechanism by which nutrient availability in the form of extracellular glucose and the enzyme O-GlcNAc Transferase (OGT), whose activity depends on glucose availability, regulates mitochondrial motility in neurons. Activation of OGT diminishes mitochondrial motility. We establish the mitochondrial motor-adaptor protein Milton as a required substrate for OGT to arrest mitochondrial motility by mapping and mutating the key O-GlcNAcylated serine residues. We find that the GlcNAcylation state of Milton is altered by extracellular glucose and that OGT alters mitochondrial motility in vivo. Our findings suggest that, by dynamically regulating Milton GlcNAcylation, OGT tailors mitochondrial dynamics in neurons based on nutrient availability.