The discovery of labile methyl esters on proliferating cell nuclear antigen by MS/MS

Abstract

The post-translational modification of proliferating cell nuclear antigen (PCNA) has been implicated in modulating its function for over 20 years. With multiple interacting partners, PCNA is involved in processes ranging from DNA replication and repair to cell cycle control and apoptosis. The ability of PCNA to distinguish between specific binding partners in different tasks is currently of intense interest, and several post-translational modifications have been reported to modulate its function. Unfortunately, these reports have produced contradictory information on the type(s) of modification present on the molecule. Here we report a detailed structural analysis of a single acidic PCNA isoform, cancer-specific polyferating nuclear anitgen (csPCNA), isolated from breast cancer cells by 2D-PAGE and LC-MS/MS. With this approach we fully characterized the csPCNA isoform and confidently identified a single post-translational modification, methyl esterification. Interestingly, the methyl esters consistently localized to 15 specific glutamic and aspartic acid residues of csPCNA. The methyl esterification of csPCNA represents a novel type of post-translational modification in mammalian cells that could ultimately hold the key towards unlocking its diverse functions.

A cancer-associated PCNA expressed in breast cancer has implications as a potential biomarker

“…We recently reported on the mass spectral analysis of the caPCNA isoform (30). This analysis did not identify the presence of acetylated, ribosylated, or phosphorylated forms of PCNA.…”

“…We have very recently described the first mass spectral analysis for any isoform of PCNA (30) and detailed the posttranslational modifications present on the caPCNA isoform. A comparison of the caPCNA posttranslational modifications with that of nmPCNA should give us insights into not only the altered posttranslational modification pathway(s) responsible for the expression of these different isoforms in breast cancer cells but also the sites of modification on the protein that may differ between the two isoforms.…”

Validating the disruption of proliferating cell nuclear antigen interactions in the development of targeted cancer therapeutics

“…12 The acidic isoform associated with cancer (caPCNA) is not the result of genetic mutations or alternate splicing, but is the result of differential post-translational modification within the PCNA molecule. 13 The specificity of…”

Human hepatocellular carcinoma expresses specific PCNA isoforms: an in vivo and in vitro evaluation

“…It is conceivable that, in malignant cells, PCNA structure could undergo modulation in the face of the increased DNA-repair activity required by a high genomic instability. Indeed, in breast cancer a specific PCNA isoform was described, termed cancer-specific PCNA, 23,33,34 although its functional role is not yet known.…”

“…Several reports have suggested the existence of PCNA modifications, 9,[16][17][18] ranging from phosphorylation, 10,19,20 not confirmed by others' data, 21,22 to acetylation 22 and, recently, to methyl esterification. 23 Additionally, PCNA mono and poly-ubiquitylation in response to DNA damage and sumoylation in the absence of damage was described. [24][25][26][27][28][29] Other than forming a homotrimeric structure, PCNA also has been found to form double homotrimeric complexes.…”