MassIVEapplication/xmlftp://massive-ftp.ucsd.edu/v09/MSV000097912/primaryOK200ProteomicsSteven G. ClarkeOrbitrap Fusion LumosHomo Sapiens (ncbitaxon:9606)https://massive.ucsd.edu/ProteoSAFe/dataset.jsp?task=d23a11572f36464bb8137e5eecc318f8clarke@mbi.ucla.eduDepartment of Chemistry and Biochemistry, University of California, Los Angeles, Los Angeles, CA 90095MassIVE13MS:1002864 - No post-translational-modifications are included in the identified peptides of this datasetA major type of spontaneous protein damage that accumulates with age is the formation of kinked polypeptide chains with L-isoaspartyl residues. Mitigating this damage is necessary for maintaining proteome stability and prolonging organismal survival. Although repair through methylation by PCMT1 has been previously shown to suppress L-isoaspartyl accumulation, we provide an additional mechanism for L-isoaspartyl maintenance through PCMTD1, a cullin-RING ligase (CRL). We combined cryo-EM, native mass spectrometry, and biochemical assays to provide insight on how the assembly and architecture of human PCMTD1 in the context of a CRL complex fulfills this alternative mechanism. We show that the PCMTD1 CRL complex specifically binds L-isoaspartyl residues when bound to AdoMet. This work provides evidence for a growing class of E3 ubiquitin ligases that recognizes spontaneous covalent modifications as potential substrates for ubiquitylation and subsequent proteasomal degradation.Structural basis for L-isoaspartyl-containing protein recognition by the human PCMTD1 cullin-RING E3 ubiquitin ligase.Pang Eric Z EZ, Zhao Boyu B, Flowers Cameron C, Oroudjeva Elizabeth E, Winter Jasmine B JB, Pandey Vijaya V, Sawaya Michael R MR, Wohlschlegel James J, Loo Joseph A JA, Rodriguez Jose A JA, Clarke Steven G SGfalseStructural basis for L-isoaspartyl-containing protein recognition by the PCMTD1 cullin-RING E3 ubiquitin ligaseLC/MS identification of proteins co-immunoprecipitated with PCMTD1 and PCMTD1 P243 F247 stably expressed in FlpIn 293 cells.Mon May 19 18:11:00 BST 2025MSV00009791240975169