Pang, E. Z. | bioRxiv Science

bioRxiv2025

Structural basis for L-isoaspartyl-containing protein recognition by the PCMTD1 cullin-RING E3 ubiquitin ligase

A 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. While 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 PCMTD1 in the context of a CRL complex fulfils 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 recognize spontaneous covalent modifications as potential substrates for ubiquitylation and subsequent proteasomal degradation. eTOC BlurbLimiting the accrual of L-isoaspartyl damaged proteins is essential during aging. While this is thought to be mediated solely by the repair activity of the protein, PCMT1, Pang et al. now demonstrate that a related protein, PCMTD1, functions as a cullin-RING ligase to selectively target L-isoaspartyl-damaged substrates for potential regulation by the ubiquitylation-proteosomal system. HighlightsO_LIAtomic cryo-EM structure of CRL5-PCMTD1 determined C_LIO_LIArchitecture of PCMTD1 when complexed as a CRL supports ubiquitylation activity C_LIO_LIPCMTD1 recognizes L-isoaspartyl residues as a recruitment motif for potential CRL activity C_LIO_LIRecognition of L-isoaspartyl residues is dependent on cofactor engagement C_LI

biochemistry↗

bioRxiv2024

PEX1G843D remains functional in peroxisome biogenesis but is rapidly degraded by the proteasome

The PEX1/PEX6 AAA-ATPase is required for the biogenesis and maintenance of peroxisomes. Mutations in HsPEX1 and HsPEX6 disrupt peroxisomal matrix protein import and are the leading cause of Peroxisome Biogenesis Disorders (PBDs). The most common disease-causing mutation in PEX1 is the HsPEX1G843D allele, which results in a reduction of peroxisomal protein import. Here we demonstrate that in vitro the homologous yeast mutant, ScPex1G700D, reduces the stability of Pex1s active D2 ATPase domain and impairs assembly with Pex6, but can still form an active AAA-ATPase motor. In vivo, ScPex1G700D exhibits only a slight defect in peroxisome import. We generated model human HsPEX1G843D cell lines and show that PEX1G843D is rapidly degraded by the proteasome, but that induced overexpression of PEX1G843D can restore peroxisome import. Additionally, we found that the G843D mutation reduces PEX1s affinity for PEX6, and that impaired assembly is sufficient to induce degradation of PEX1WT. Lastly, we found that fusing a deubiquitinase to PEX1G843D significantly hinders its degradation in mammalian cells. Altogether, our findings suggest a novel regulatory mechanism for PEX1/PEX6 hexamer assembly and highlight the potential of protein stabilization as a therapeutic strategy for PBDs arising from the G843D mutation and other PEX1 hypomorphs.

biochemistry↗