利用工程化PCMT1解码SCLC中ELAVL4的异天冬氨酰化：一种揭示免疫触发因素的策略

The 5-year survival rate for SCLC patients is only 9%-new therapies are urgently needed. Approximately 15% of SCLC patients develop a low-titer immune response against the neuronal protein embryonic lethal abnormal vision-like 4 (ELAVL4), exhibiting significantly improved survival. In the lung, ELAVL4 is uniquely expressed in pulmonary neuroendocrine cells, the primary cell of origin of SCLC. Prior research in our lab suggests that the anti-ELAVL4 immune response originates from an immunogenic type of post-translational damage called isoaspartylation. We previously mapped the isoaspartylation of ELAVL4 to the 38-amino acid N-terminal unstructured region, upstream of the RNA recognition motif domain (RRM1). Isoaspartylation can occur at asparagine and aspartic acid residues, most commonly when they are located in a flexible protein region and followed by a small amino acid (glycine, serine, or histidine). The asparagine or aspartate side chain reacts with the main chain, forming a cyclic succinimide intermediate that, in 70% of cases, resolves to an isoaspartate (isoAsp), thereby kinking the peptide backbone. Isoaspartylation is repaired by the enzyme protein-L-isoaspartate (D-aspartate) O-methyltransferase (PCMT1) in vivo. PCMT1 deficiency is invariably fatal in mice, illustrating the essential nature of this repair enzyme. PCMT1 utilizes the methyl donor S-adenosylmethionine (SAM) to initiate repair, producing S-adenosylhomocysteine (SAH), which can be quantified via ELISA and bioluminescent assays. Mapping the isoaspartylation site(s) in ELAVL4 is essential, as this will help identify the antigenic epitope used to design future targeted immunotherapies for SCLC patients. However, mapping of isoaspartylation sites is challenging due to minimal molecular weight differences between asparagine, aspartate, and isoaspartate residues. In the past, tritiated SAM has been used in conjunction with PCMT1 to label proteins and detect isoaspartylation. Here, we investigate whether PCMT1 can be utilized in conjunction with mass spectrometry to methylate and detect isoaspartylation in ELAV4. This method is site-specific and avoids the use of radioactivity. We will also assess substrate binding kinetics using a Bruker heliX biosensor. Improved detection and mapping of isoaspartylation sites in ELAVL4 can help identify therapeutic targets that leverage the anti-isoaspartylated ELAVL4 response for new SCLC treatments. Supported by the Robert E. and May R. Wright Foundation Transformative Cancer Research Grant, USC's Undergraduate Research Associates Program, and the Norris Comprehensive Cancer Center core grant, NIH/NCI P30CA014089. DAV and IAO are members of CaRE2 the Cancer Research Education and Engagement Health Center, supported by NIH/NCI grants U54CA233396, U54CA233444, and U54233465.