Isolation of Mammalian 26S Proteasomes and p97/VCP Complexes Using the Ubiquitin-like Domain from HHR23B Reveals Novel Proteasome-Associated Proteins

Recent studies, mainly in yeast, have identified various cofactors that associate with the 26S proteasome and appear to influence its function. To identify these proteins in different cells and physiological states, we developed a method to gently and rapidly isolate 26S proteasomes and associated proteins without the need for genetic modifications of the proteasome. This method is based on the affinity of this complex for the ubiquitin-like (UBL) domain of hHR23B and elution with a competing polypeptide containing a ubiquitin-interacting motif. Associated with 26S proteasomes from rat muscle were a variety of known proteasome-interacting proteins, activators, and ubiquitin conjugates. In addition, we identified over 40 proteins not previously known to associate with the 26S proteasome, some of which were tightly associated with the proteasome in a substoichiometric fashion, e.g., the deubiquitinating enzymes USP5/isopeptidase T and USP7/HAUSP and the ubiquitin ligases ARF-BP1/HUWE1 and p600/UBR4. By altering buffer conditions, we also purified by this approach complexes of the ATPase p97/VCP associated with its adaptor proteins Ufd1-Npl4, p47, SAKS1, and FAF1, all of which contain ubiquitin-binding motifs. These complexes were isolated with ubiquitin conjugates bound and were not previously known to bind to the UBL domain of hHR23B. These various UBL-interacting proteins, dubbed the UBL interactome, represent a network of proteins that function together in ubiquitin-dependent proteolysis, and the UBL method offers many advantages for studies of the diversity, functions, and regulation of 26S proteasomes and p97 complexes under different conditions

Isolation of Mammalian 26S Proteasomes and p97/VCP Complexes Using the Ubiquitin-like Domain from HHR23B Reveals Novel Proteasome-Associated Proteins

Recent studies, mainly in yeast, have identified various cofactors that associate with the 26S proteasome and appear to influence its function. To identify these proteins in different cells and physiological states, we developed a method to gently and rapidly isolate 26S proteasomes and associated proteins without the need for genetic modifications of the proteasome. This method is based on the affinity of this complex for the ubiquitin-like (UBL) domain of hHR23B and elution with a competing polypeptide containing a ubiquitin-interacting motif. Associated with 26S proteasomes from rat muscle were a variety of known proteasome-interacting proteins, activators, and ubiquitin conjugates. In addition, we identified over 40 proteins not previously known to associate with the 26S proteasome, some of which were tightly associated with the proteasome in a substoichiometric fashion, e.g., the deubiquitinating enzymes USP5/isopeptidase T and USP7/HAUSP and the ubiquitin ligases ARF-BP1/HUWE1 and p600/UBR4. By altering buffer conditions, we also purified by this approach complexes of the ATPase p97/VCP associated with its adaptor proteins Ufd1-Npl4, p47, SAKS1, and FAF1, all of which contain ubiquitin-binding motifs. These complexes were isolated with ubiquitin conjugates bound and were not previously known to bind to the UBL domain of hHR23B. These various UBL-interacting proteins, dubbed the UBL interactome, represent a network of proteins that function together in ubiquitin-dependent proteolysis, and the UBL method offers many advantages for studies of the diversity, functions, and regulation of 26S proteasomes and p97 complexes under different conditions

Isolation of Mammalian 26S Proteasomes and p97/VCP Complexes Using the Ubiquitin-like Domain from HHR23B Reveals Novel Proteasome-Associated Proteins

Recent studies, mainly in yeast, have identified various cofactors that associate with the 26S proteasome and appear to influence its function. To identify these proteins in different cells and physiological states, we developed a method to gently and rapidly isolate 26S proteasomes and associated proteins without the need for genetic modifications of the proteasome. This method is based on the affinity of this complex for the ubiquitin-like (UBL) domain of hHR23B and elution with a competing polypeptide containing a ubiquitin-interacting motif. Associated with 26S proteasomes from rat muscle were a variety of known proteasome-interacting proteins, activators, and ubiquitin conjugates. In addition, we identified over 40 proteins not previously known to associate with the 26S proteasome, some of which were tightly associated with the proteasome in a substoichiometric fashion, e.g., the deubiquitinating enzymes USP5/isopeptidase T and USP7/HAUSP and the ubiquitin ligases ARF-BP1/HUWE1 and p600/UBR4. By altering buffer conditions, we also purified by this approach complexes of the ATPase p97/VCP associated with its adaptor proteins Ufd1-Npl4, p47, SAKS1, and FAF1, all of which contain ubiquitin-binding motifs. These complexes were isolated with ubiquitin conjugates bound and were not previously known to bind to the UBL domain of hHR23B. These various UBL-interacting proteins, dubbed the UBL interactome, represent a network of proteins that function together in ubiquitin-dependent proteolysis, and the UBL method offers many advantages for studies of the diversity, functions, and regulation of 26S proteasomes and p97 complexes under different conditions

Gas-Phase Rearrangements Do Not Affect Site Localization Reliability in Phosphoproteomics Data Sets

Intramolecular transfer of phosphate during collision-induced dissociation (CID) in ion-trap mass spectrometers has recently been described. Because phosphorylation events are assigned to discrete serine, threonine, and tyrosine residues based on the presence of site-determining ions in MS/MS spectra, phosphate transfer may invalidate or confound site localization in published large-scale phosphorylation data sets. Here, we present evidence for the occurrence of this phenomenon using synthetic phosphopeptide libraries, specifically for doubly charged species. We found, however, that the extent of the transfer reaction was insufficient to cause localization of phosphorylation sites to incorrect residues. We further compared CID to electron-transfer dissociation (ETD) for site localization using synthetic libraries and a large-scale yeast phosphoproteome experiment. The agreement in site localization was >99.5 and 93%, respectively, suggesting that ETD-based site localization is no more reliable than CID. We conclude that intramolecular phosphate transfer does not affect the reliability of current or past phosphorylation data sets

Gas-Phase Rearrangements Do Not Affect Site Localization Reliability in Phosphoproteomics Data Sets

Intramolecular transfer of phosphate during collision-induced dissociation (CID) in ion-trap mass spectrometers has recently been described. Because phosphorylation events are assigned to discrete serine, threonine, and tyrosine residues based on the presence of site-determining ions in MS/MS spectra, phosphate transfer may invalidate or confound site localization in published large-scale phosphorylation data sets. Here, we present evidence for the occurrence of this phenomenon using synthetic phosphopeptide libraries, specifically for doubly charged species. We found, however, that the extent of the transfer reaction was insufficient to cause localization of phosphorylation sites to incorrect residues. We further compared CID to electron-transfer dissociation (ETD) for site localization using synthetic libraries and a large-scale yeast phosphoproteome experiment. The agreement in site localization was >99.5 and 93%, respectively, suggesting that ETD-based site localization is no more reliable than CID. We conclude that intramolecular phosphate transfer does not affect the reliability of current or past phosphorylation data sets

Gas-Phase Rearrangements Do Not Affect Site Localization Reliability in Phosphoproteomics Data Sets

Intramolecular transfer of phosphate during collision-induced dissociation (CID) in ion-trap mass spectrometers has recently been described. Because phosphorylation events are assigned to discrete serine, threonine, and tyrosine residues based on the presence of site-determining ions in MS/MS spectra, phosphate transfer may invalidate or confound site localization in published large-scale phosphorylation data sets. Here, we present evidence for the occurrence of this phenomenon using synthetic phosphopeptide libraries, specifically for doubly charged species. We found, however, that the extent of the transfer reaction was insufficient to cause localization of phosphorylation sites to incorrect residues. We further compared CID to electron-transfer dissociation (ETD) for site localization using synthetic libraries and a large-scale yeast phosphoproteome experiment. The agreement in site localization was >99.5 and 93%, respectively, suggesting that ETD-based site localization is no more reliable than CID. We conclude that intramolecular phosphate transfer does not affect the reliability of current or past phosphorylation data sets

Additional file 3: Table S2. of Tribbles ortholog NIPI-3 and bZIP transcription factor CEBP-1 regulate a Caenorhabditis elegans intestinal immune surveillance pathway

Affymetrix microarray analysis of wild type N2, nipi-3(fr4), and pmk-1(km25) gene expression. This table lists the genes differentially expressed in wild type N2 and nipi-3(fr4) (first tab) or pmk-1(km25) (second tab) animals fed control OP50 E. coli at 20 °C until the L4 stage. Expression values are the average of three independent replicates. Gene ontology terms enriched in each category are listed in the third tab. (XLSX 59 kb

Additional file 4: Table S3. of Tribbles ortholog NIPI-3 and bZIP transcription factor CEBP-1 regulate a Caenorhabditis elegans intestinal immune surveillance pathway

NanoString analysis of wild type N2 and nipi-3(fr4) gene expression. This table lists the gene expression fold changes between wild type N2 and nipi-3(fr4) animals fed P. aeruginosa PA14 or control OP50 E. coli for 6 hours (first tab) or fed E. coli expressing ToxA or BL21 control for 24 hours (second tab). Expression values are the average of three (PA14 and OP50) or two (ToxA and BL21 control) independent replicates and normalized to three control genes. Primary data are provided in Additional file 15. (XLSX 33 kb

Additional file 6: Figure S3. of Tribbles ortholog NIPI-3 and bZIP transcription factor CEBP-1 regulate a Caenorhabditis elegans intestinal immune surveillance pathway

Lifespan and gene expression analysis of nipi-3(fr4) and mutants of other immune pathways required for the ToxA response. a. qRT-PCR comparison of wild type N2, nipi-3(fr4), pmk-1(km25), zip-2(tm4248), and fshr-1(ok778) animals following exposure to P. aeruginosa PA14 or OP50 E. coli for 6 hours. Results shown are an average of two (fshr-1) or three (remaining samples) biological replicates. b. qRT-PCR comparison of wild type N2, nipi-3(fr4), and kgb-1(km21) animals following exposure to E. coli expressing ToxA or the BL21 control for 24 hours. Results shown are an average of two biological replicates. For a and b, results are normalized to the value of wild type worms on control E. coli for the given gene. Error bars represent SEM. *P < 0.05 (Student’s t-test) when compared to the corresponding wild type animals. c. Lifespans of wild type N2, pmk-1(km25), zip-2(tm4248), and nipi-3(RNAi) fed E. coli expressing ToxA. Number of animals scored for each condition was ≥ 55 (257 total). This is a representative experiment of three independent experiments. Primary data for panels a and b are provided in Additional file 15. (PDF 463 kb

It Makes Me Spit: The Public and Newspaper Reaction to the UK Governmentr's Threat to Suppress the Daily Mirror

cebp-1 acts in the intestine and affects immune gene expression. a. Lifespans of MGH167 (gut RNAi) animals grown on equal mixtures of cebp-1 RNAi and L4440 vector control; nipi-3 RNAi and L4440 vector control; cebp-1 and nipi-3 RNAi; or L4440 vector control alone to the L4 stage. Animals were then transferred to E. coli expressing ToxA. Note that the mixed nipi-3 RNAi showed less ToxA susceptibility than undiluted nipi-3 RNAi (Fig. S1b). P = 0.95 (log-rank test) and 0.4 (Wilcoxon test) for nipi-3, cebp-1 versus cebp-1 RNAi; P = 0.089 (log-rank test) and 0.0004 (Wilcoxon test) for nipi-3 versus cebp-1 RNA. Number of animals scored for each condition was > 65 (426 total). This is a representative experiment of two independent experiments. b. qRT-PCR comparison of the indicated strains exposed to E. coli expressing ToxA for 24 hours. Results shown are an average of two biological replicates and are normalized to the corresponding wild type ToxA value. Error bars represent SEM. nipi-3 refers to nipi-3(fr4). Primary data for panel b are provided in Additional file 15. (PDF 206 kb