benzophenone 4 carboxamidocysteine methanethiosulfonate bpmts  (Toronto Research Chemicals)

Journal: EMBO Reports

Article Title: DPP8/9 processing of human AK2 unmasks an IAP binding motif

doi: 10.1038/s44319-025-00455-z

Figure Lengend Snippet: ( A ) A bioinformatic screen on human genome (35589 unique consensus coding sequences) was performed revealing 3465 proteins with potential DPP8/9 cleavage sites of which 129 contain an IBM behind the cleavage site (list, see Appendix Fig. S ). ( B ) Model of IBM unmasking by DPP8/9 processing. DPP8/9 processing results in the unmasking of an IBM which facilitates increased degradation of the protein. There appears to be an entire class of proteins with similar features (DPP8/9 cleavage site followed by an IBM), most of them localized to the cytosol. Among them was also the translation initiation factor EIF2A. Its different N-terminal variants are displayed on the right. ( C ) Strategy to identify N-termini of EIF2A in HEK293 cells. Cells were incubated with the proteasome inhibitor MG132 for 6 h. After lysis, endogenous EIF2A was immunoprecipitated and the resulting precipitate analyzed by mass spectrometry. ( D ) In intact cells, four different N-terminal peptides of EIF2A were identified by MS analysis: processed acetylated and free NH 2 -STPLLTV R , NH 2 -APSTPLLTRV R and NH 2 -MAPSTPLLTRV R . EIF2A was immunoprecipitated from HEK293 WT and DPP8/9 KO cells treated with MG132 6 h before lysis and precipitates then analyzed by MS. The raw intensities of the four N-terminal peptides comparing HEK293 WT with DPP8/9 KO cells are plotted as bar charts for each peptide. For “peptides w/o N-terminus” mean intensities of non-N-Terminal peptides (WT n = 54; KO n = 55) of EIF2A were calculated and plotted. Experimental replicates n = 2. ( E ) Peptide constructs used for interaction screen. Various peptides containing amino acid residues representing different EIF2A termini were used for an interaction screen using HeLa cell lysate. ( F ) EIF2A processed by MAP and DPP8/9 but not unprocessed EIF2A interacts with IAPs. Peptides with the respective N-terminal peptide of EIF2A were incubated with HeLa cell lysates. Interaction partners were identified using MS. Pairwise, two-sided Student’s t test was used for statistical testing. n = 3 biological replicates. ( G ) EIF2A processed by MAP and DPP8/9 but not MAP-processed AK2 interacts with IAPs. Peptides with the respective N-terminal peptide of EIF2A were incubated with HeLa cell lysates. Interaction partners were identified using MS. Pairwise, two-sided Student’s t test was used for statistical testing. n = 3 biological replicates. ( H ) Acetylation of the N-terminus of EIF2A inhibits the interaction with IAPs. Either acetylated or non-acetylated N-terminal EIF2A peptides were incubated with HeLa cell lysates. Interaction partners were identified using MS. ( I ) Depletion of NatA levels destabilizes EIF2A2. The levels of the catalytic domain of NatA were depleted using siRNA in HeLa cells. Cell lysates were analyzed via SDS-PAGE and immunoblot. n = 3 biological replicates. ( J ) XIAP ubiquitinates the DPP8/9-processed free N-terminus of EIF2A in vitro. The experiment was performed as explained in Fig. . The indicated N-terminal peptides of EIF2A were subjected to an in vitro ubiquitination assay using purified GST-XIAP. An example of an immunoblot is shown in the left panel. In the right panel, the ratio of −/+ XIAP of quantified immunoblots ( n = 2) for each peptide normalized on streptavidin amounts is plotted. ( K ) Model. Different strategies exist to mask IBMs in cells. Unmasked IBMs result in rapid degradation of the protein and potentially sensitization of cells toward apoptosis. Equipping the pro-proteins SMAC/DIABLO and HtrA2 with bpMTS for import into the IMS blocks the IBM during import into the IMS. Cleavage of the bpMTS inside mitochondria unmasks the IBM, which is however not accessible to cytosolic IAPs until release of the factors is initiated during apoptosis induction. Likewise, proteins are masked by DPP8/9-cleavable peptides until these peptidases process the proteins. Lastly, N-terminal acetylation of exposed IBMs prevents their binding to IAPs. .

Article Snippet: We complemented this approach with another experiment in which we constructed an AK2 variant that lacked the first three amino acids (mimicking the N-Terminus after MAP and DPP8/9 cleavage) and was equipped with the SMAC bpMTS (SMAC MTS ) (Appendix Fig. S ).

Techniques: Incubation, Lysis, Immunoprecipitation, Mass Spectrometry, Construct, SDS Page, Western Blot, In Vitro, Ubiquitin Proteomics, Purification, Binding Assay