Mass Spectrometry Analysis of Proteome-Wide Proteolytic Post-Translational Degradation of Proteins

Abstract

Protein proteolytic degradation is an essential component to proper cell function and its life cycle. Here, we study the protein degradation in yeast Saccharomyces cerevisiae cells on a proteome-wide scale by detection of the intermediate peptides produced from the intracellular degradation of proteins using sequencing-based tandem mass spectrometry. By tracing the detected ~1,100 peptides and their 200 protein substrate origins we obtain evidence for new insights into the proteome-wide protein selective degradation in yeast cells. This evidences shows that the yeast cytoplasm is the largest pool for the degradation of proteins with both biochemical and geometric specificities, while the yeast nucleus seems to be a proteolysis-inert organelle under the condition studied. Yeast V-ATPase subunits appear to be degraded during their disassembly, and yeast mitochondrial proteins functioning as precursors, transport carriers and gates are preferentially degraded. Ubiquitylation may be unnecessary for the proteasomal degradation of yeast cytoplasmic regulatory and enzyme proteins according to our observations. This study shows that the intracellular peptides are informational targets for directly probing the protein degradation-involved molecular mechanisms and cell biology processes.Protein degradation is a post-translational modification necessary for protein quality control and cell survival. In eukaryotic cells degradation of proteins is primarily carried out in either the proteasomes or lysosomes. Proteasomes, 1,2 are responsible for protein quality control by breaking down misfolded proteins, 3 and they have a well-defined geometric proteolysiscatalytic center 4,5 that breaks down the proteins to produce peptides with specific lengths. 6,7 Ubiquitylation 7,8 of proteins is the typical initial step for protein degradation through the proteasomes (note: ubiquitylation also functions for non-proteolytic purposes 10 ). Resultant peptides from proteasomal degradation can then be further cleaved into amino acids by cytosolic aminopeptidases. 11 Lysosomes, on the other hand, are membrane bound organellelike structures containing multiple proteases that degrade protein aggregates and membrane proteins. 3,12 Proteins to-be-degraded are transported to the lysosome through e.g., the ATGs/ signaling/regulating systems 13,14 and the following proteolyses are catalyzed by endoproteinases 15,16 that are matured and activated within the lysosome. The resultant peptides continue to break down into amino acids by the lysosomal aminopeptidases and carboxypeptidases, 16,17 and the products (e.g., amino acids and others such as sugars, cholesterols) are then transported back out into the cytosol by the solute transporters in the Requests for materials should be addressed to Yufeng Shen (E-mail: yufeng.shen@pnl.gov) or Richard D. Smith (E-mail: dick.smith@pnl.gov). NIH Public Access NIH-PA Author ManuscriptNIH-PA Author Manuscript NIH-PA Author Manuscript lysosomal membrane and can then be recycled as nutrients making this de...

Disaggregation of Amylin Aggregate by Novel Conformationally Restricted Aminobenzoic Acid containing α/β and α/γ Hybrid Peptidomimetics

“…Furthermore, 2-aminobenzoic acid derivatives have been used as transthyretin amyloid fibril25 and amyloid β peptide26 aggregation inhibitors. We hypothesized that the hybrid peptides containing β, γ and δ aminobenzoic acids would be more stable towards proteolytic degradation2728, and thus a better candidate that inhibit/disrupt hIAPP aggregation. We wanted to find better lead molecules for drug design against T2DM by a minimalistic design approach and incorporation of conformational restriction using differently substituted amino benzoic acids.…”

Protein Identification Using Top-Down Spectra

“…• In addition, MASCOT, SEQUEST, and OMSSA (16,26,27) have been used for top-down protein identification.…”

De Novo Sequencing of Unique Sequence Tags for Discovery of Post-Translational Modifications of Proteins

“…The FT-MS/MS data set used in this work was that used for a previous study on the intracellular proteolytic degradation of yeast proteins 2. Briefly, a lysate was quickly extracted from yeast Saccharomyces cerevisiae cells with the use of pressure cycling technology and in the presence of a protease inhibitor cocktail (Roche, Indianapolis, IN).…”

“…Sequences are determined to be UStags when the accurately measured consecutive fragments reveal these sequences to be unique in the genome for single proteins. The UStags reported1,2 are assigned for the candidates that have the top closest spectral similarities to the MS/MS measurement (e.g., candidates ranked from Sequest). Advantage of such a database search–UStag approach is that it produces sequence identities with extremely low false discovery rates for peptides/polypeptides having a large range of lengths and with various amino acid termini 1,2.…”