Seamless multiresolution isosurfaces using wavelets

Data sets that are being produced by today's simulations, such as the ones generated by DOE's ASCI program, are too large for real-time exploration and visualization. Therefore, new methods of visualizing these data sets need to be investigated. The authors present a method that combines isosurface representations of different resolutions into a seamless solution, virtually free of cracks and overlaps. The solution combines existing isosurface generation algorithms and wavelet theory to produce a real-time solution to multiple-resolution isosurfaces

Rapid and deep-scale ubiquitylation profiling for biology and translational research

Protein ubiquitylation is involved in a plethora of cellular processes. While antibodies directed at ubiquitin remnants (K-ɛ-GG) have improved the ability to monitor ubiquitylation using mass spectrometry, methods for highly multiplexed measurement of ubiquitylation in tissues and primary cells using sub-milligram amounts of sample remains a challenge. Here, we present a highly sensitive, rapid and multiplexed protocol termed UbiFast for quantifying ~10,000 ubiquitylation sites from as little as 500 μg peptide per sample from cells or tissue in a TMT10plex in ca. 5 h. High-field Asymmetric Waveform Ion Mobility Spectrometry (FAIMS) is used to improve quantitative accuracy for posttranslational modification analysis. We use the approach to rediscover substrates of the E3 ligase targeting drug lenalidomide and to identify proteins modulated by ubiquitylation in models of basal and luminal human breast cancer. The sensitivity and speed of the UbiFast method makes it suitable for large-scale studies in primary tissue samples

Identification of Combinatorial Patterns of Post-Translational Modifications on Individual Histones in the Mouse Brain

Post-translational modifications (PTMs) of proteins are biochemical processes required for cellular functions and signalling that occur in every sub-cellular compartment. Multiple protein PTMs exist, and are established by specific enzymes that can act in basal conditions and upon cellular activity. In the nucleus, histone proteins are subjected to numerous PTMs that together form a histone code that contributes to regulate transcriptional activity and gene expression. Despite their importance however, histone PTMs have remained poorly characterised in most tissues, in particular the brain where they are thought to be required for complex functions such as learning and memory formation. Here, we report the comprehensive identification of histone PTMs, of their combinatorial patterns, and of the rules that govern these patterns in the adult mouse brain. Based on liquid chromatography, electron transfer, and collision-induced dissociation mass spectrometry, we generated a dataset containing a total of 10,646 peptides from H1, H2A, H2B, H3, H4, and variants in the adult brain. 1475 of these peptides carried one or more PTMs, including 141 unique sites and a total of 58 novel sites not described before. We observed that these PTMs are not only classical modifications such as serine/threonine (Ser/Thr) phosphorylation, lysine (Lys) acetylation, and Lys/arginine (Arg) methylation, but also include several atypical modifications such as Ser/Thr acetylation, and Lys butyrylation, crotonylation, and propionylation. Using synthetic peptides, we validated the presence of these atypical novel PTMs in the mouse brain. The application of data-mining algorithms further revealed that histone PTMs occur in specific combinations with different ratios. Overall, the present data newly identify a specific histone code in the mouse brain and reveal its level of complexity, suggesting its potential relevance for higher-order brain functions

Targeting of EGFR by a combination of antibodies mediates unconventional EGFR trafficking and degradation

Antibody combinations targeting cell surface receptors are a new modality of cancer therapy. The trafficking and signalling mechanisms regulated by such therapeutics are not fully understood but could underlie differential tumour responses. We explored EGFR trafficking upon treatment with the antibody combination Sym004 which has shown promise clinically. Sym004 promoted EGFR endocytosis distinctly from EGF: it was asynchronous, not accompanied by canonical signalling events and involved EGFR clustering within detergent-insoluble plasma mebrane-associated tubules. Sym004 induced lysosomal degradation independently of EGFR ubiquitylation but dependent upon Hrs/Tsg101 that are required for the formation of intraluminal vesicles (ILVs) within late endosomes. We propose Sym004 cross-links EGFR physically triggering EGFR endocytosis and incorporation onto ILVs and so Sym004 sensitivity correlates with EGFR numbers available for binding, rather than specific signalling events. Consistently Sym004 efficacy and potentiation of cisplatin responses correlated with EGFR surface expression in head and neck cancer cells. These findings will have implications in understanding the mode of action of this new class of cancer therapeutics

Capzimin is a potent and specific inhibitor of proteasome isopeptidase Rpn11

The proteasome is a vital cellular machine that maintains protein homeostasis, which is of particular importance in multiple myeloma and possibly other cancers. Targeting of proteasome 20S peptidase activity with bortezomib and carfilzomib has been widely used to treat myeloma. However, not all patients respond to these compounds, and those who do eventually suffer relapse. Therefore, there is an urgent and unmet need to develop new drugs that target proteostasis through different mechanisms. We identified quinoline-8-thiol (8TQ) as a first-in-class inhibitor of the proteasome 19S subunit Rpn11. A derivative of 8TQ, capzimin, shows >5-fold selectivity for Rpn11 over the related JAMM proteases and >2 logs selectivity over several other metalloenzymes. Capzimin stabilized proteasome substrates, induced an unfolded protein response, and blocked proliferation of cancer cells, including those resistant to bortezomib. Proteomic analysis revealed that capzimin stabilized a subset of polyubiquitinated substrates. Identification of capzimin offers an alternative path to develop proteasome inhibitors for cancer therapy

Large-scale identification of ubiquitination sites by mass spectrometry.

Ubiquitination is essential for the regulation of cellular protein homeostasis. It also has a central role in numerous signaling events. Recent advances in the production and availability of antibodies that recognize the Lys-{epsilon}-Gly-Gly (K-{epsilon}-GG) remnant produced by trypsin digestion of proteins having ubiquitinated lysine side chains have markedly improved the ability to enrich and detect endogenous ubiquitination sites by mass spectrometry (MS). The following protocol describes the steps required to complete a large-scale ubiquitin experiment for the detection of tens of thousands of distinct ubiquitination sites from cell lines or tissue samples. Specifically, we present detailed, step-by-step instructions for sample preparation, off-line fractionation by reversed-phase chromatography at pH 10, immobilization of an antibody specific to K-{epsilon}-GG to beads by chemical cross-linking, enrichment of ubiquitinated peptides using these antibodies and proteomic analysis of enriched samples by LC-tandem MS (MS/MS). Relative quantification can be achieved by performing stable isotope labeling by amino acids in cell culture (SILAC) labeling of cells. After cell or tissue samples have been prepared for lysis, the described protocol can be completed in ~5 d

Refined preparation and use of anti-diglycine remnant (K-ε-GG) antibody enables routine quantification of 10,000s of ubiquitination sites in single proteomics experiments

Detection of endogenous ubiquitination sites by mass spectrometry has dramatically improved with the commercialization of anti-di-glycine remnant (K-{epsilon}-GG) antibodies. Here, we describe a number of improvements to the K-{epsilon}-GG enrichment workflow, including optimized antibody and peptide input requirements, antibody cross-linking, and improved off-line fractionation prior to enrichment. This refined and practical workflow enables routine identification and quantification of ~20,000 distinct endogenous ubiquitination sites in a single SILAC experiment using moderate amounts of protein input