In-depth proteomic analysis of proteasome inhibitors bortezomib, carfilzomib and MG132 reveals that mortality factor 4-like 1 (MORF4L1) protein ubiquitylation is negatively impacted.

Proteasome inhibitors are an important class of chemotherapeutic drugs. In this study, we performed a large-scale ubiquitylome analysis of the three proteasome inhibitors MG132, bortezomib and carfilzomib. Although carfilzomib is currently being used for the treatment of multiple myeloma, it has not yet been subjected to a global ubiquitylome analysis.

A conserved quality-control pathway that mediates degradation of unassembled ribosomal proteins.

Overproduced yeast ribosomal protein (RP) Rpl26 fails to assemble into ribosomes and is degraded in the nucleus/nucleolus by a ubiquitin-proteasome system quality control pathway comprising the E2 enzymes Ubc4/Ubc5 and the ubiquitin ligase Tom1. tom1 cells show reduced ubiquitination of multiple RPs, exceptional accumulation of detergent-insoluble proteins including multiple RPs, and hypersensitivity to imbalances in production of RPs and rRNA, indicative of a profound perturbation to proteostasis. Tom1 directly ubiquitinates unassembled RPs primarily via residues that are concealed in mature ribosomes.

A Targeted MRM Approach for Tempo-Spatial Proteomics Analyses.

When deciding to perform a quantitative proteomics analysis, selectivity, sensitivity, and reproducibility are important criteria to consider. The use of multiple reaction monitoring (MRM) has emerged as a powerful proteomics technique in that regard since it avoids many of the problems typically observed in discovery-based analyses. A prerequisite for such a targeted approach is that the protein targets are known, either as a result of previous global proteomics experiments or because a specific hypothesis is to be tested.

ETD Outperforms CID and HCD in the Analysis of the Ubiquitylated Proteome.

Comprehensive analysis of the ubiquitylome is a prerequisite to fully understand the regulatory role of ubiquitylation. However, the impact of key mass spectrometry parameters on ubiquitylome analyses has not been fully explored. In this study, we show that using electron transfer dissociation (ETD) fragmentation, either exclusively or as part of a decision tree method, leads to ca.

Recent advances in defining the ubiquitylome.

Ubiquitin is a small 8.5 kDa protein that is conjugated to a target protein in a concerted three step enzymatic process. Ubiquitin addition can drastically affect function or target the modified protein for degradation.

Yeast ribosomal/cytochrome c SET domain methyltransferase subfamily: identification of Rpl23ab methylation sites and recognition motifs.

Ribosomal protein L23ab is specifically dimethylated at two distinct sites by the SET domain-containing enzyme Rkm1 in the yeast Saccharomyces cerevisiae. Using liquid column chromatography with electrospray-ionization mass spectrometry, we determined that Rpl23ab purified from the Deltarkm1 deletion strain demonstrated a loss in mass of approximately 56 Da when compared with Rpl23ab purified from the wild type strain. When Rpl23ab was proteolyzed, using proteinase ArgC or CNBr, and the peptides derived were analyzed by tandem mass spectrometry, no sites of methylation were found in Rpl23ab purified from the Deltarkm1 deletion strain, whereas two sites of dimethylation were observed in the wild type strain at lysine residues 105 and 109.

A novel SET domain methyltransferase in yeast: Rkm2-dependent trimethylation of ribosomal protein L12ab at lysine 10.

The ribosomal protein L12ab (Rpl12ab) in Saccharomyces cerevisiae is modified by methylation at both arginine and lysine residues. Although the enzyme responsible for the modification reaction at arginine 66 has been identified (Rmt2), the enzyme(s) responsible for the lysine modification(s) has not been found, and the site(s) of methylation has not been determined. Here we demonstrate, using a combination of mass spectrometry and labeling assays, that the yeast gene YDR198c encodes the enzyme responsible for the predominant epsilon-trimethylation at lysine 10 in Rpl12ab.

A novel SET domain methyltransferase modifies ribosomal protein Rpl23ab in yeast.

In vivo studies have shown that the ribosomal large subunit protein L23a (Rpl23ab) in Saccharomyces cerevisiae is methylated at lysine residues. However, the gene encoding the methyltransferase responsible for the modification has not been identified. We show here that the yeast YPL208w gene product, a member of the SET domain family of methyltransferases, catalyzes the reaction, and we have now designated it Rkm1 (ribosomal lysine (K) methyltransferase 1).