Natural isoaspartyl protein modification of ZAP70 alters T cell responses in lupus

Abstract

Protein posttranslational modifications (PTMs) arise in a number of normal cellular biological pathways and in response to pathology caused by inflammation and/or infection. Indeed, a number of PTMs have been identified and linked to specific autoimmune responses and metabolic pathways. One particular PTM, termed isoaspartyl (isoAsp or isoD) modification, is among the most common spontaneous PTM occurring at physiological pH and temperature. Herein, we demonstrate that isoAsp modifications arise within the ZAP70 protein tyrosine kinase upon T-cell antigen receptor (TCR) engagement. The enzyme protein L-isoaspartate O-methyltransferase (PCMT1, or PIMT, EC 2.1.1.77) evolved to repair isoaspartyl modifications in cells. In this regard, we observe that increased levels of isoAsp modification that arise under oxidative stress are correlated with reduced PIMT activity in patients with systemic lupus erythematosus (SLE). PIMT deficiency leads to T cell hyper-proliferation and hyper-phosphorylation through ZAP70 signaling. We demonstrate that inducing the overexpression of PIMT can correct the hyper-responsive phenotype in lupus T cells. Our studies reveal a phenotypic role of isoAsp modification and phosphorylation of ZAP70 in lupus T cell autoimmunity and provide a potential therapeutic target through the repair of isoAsp modification.

Keywords:

• posttranslational modifications
• isoaspartyl modification
• protein L-isoaspartate O-methyltransferase
• TCR signaling
• systemic lupus erythematosus

Acknowledgments

We thank Renelle Gee and Anthony Ferrandino for technical assistant for the study; Susan Kaech (Yale School of Medicine, Connecticut) for providing the MSCV and pEco vector; Joe Craft and Ping Zhou (Yale School of Medicine, Connecticut) for providing mPCC transgenic mice; Min Sun Shin and Naeun Lee for purifying PBMC from human samples; Wuyi Meng (Chemical and Biophysical Instrumentation Center, Yale University) for critical discussion for ZAP70 crystal structure; and Yanhong Deng (Yale Center for Analytical Sciences, Yale School of Public Health) for statistical help of correlation analysis.

Author contributions

M.L.Y. designed and performed the experiments and wrote the manuscript. T.T.L and J.K. did the mass spectrometry experiments and interpreted the results. I.K. collected human samples of SLE patients and healthy donors/subjects and provided advice for handling human samples. Z.S.Z. contributed the critical discussion for PIMT-mediated ¹⁸O-labeling of isoaspartic acid by mass spectrometry. S.G.C. contributed conceptual expertise, provided inputs into the design of the study, and edited the paper. M.J.M. supervised all the experiments and edited the manuscript.

Disclosure statement

The authors declare no competing financial interests.

Data availability statement

The data that support the findings of this study are available from the corresponding author upon reasonable request.

Additional information

Funding

This work was supported by National Institutes of Health Grant (RO1 AI-48120) to M.J.M.