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Abstract
Background T cell responses are known to be amplified in SLE, via TCR pathways of signal transduction. In particular, increases in intracellular tyrosine phosphorylation, calcium flux and actin polymerization are among the abnormal responses found in SLE T cells. An imbalance of how T cells signal through both Syk and ZAP70 shape the activation and cytokine responses in human SLE. The present study examines posttranslational protein modifications (PTMs) that arise in the context of SLE inflammation. PTMs are known to alter both immune tolerance to self proteins (such as citrulline PTMs that are diagnostic in RA) as well as intracellular metabolic and signalling pathways. In particular, isoaspartyl (isoAsp) modification is one intracellular PTM previously demonstrated to be increased by cellular stress and inflammation. The present study examined T cell biology that is altered by PTMs in lupus.
Materials and methods Isoaspartyl PTMs were characterised in lymphocytes from both human SLE and in murine models. We specifically examined ZAP70 for PTMs to determine effects on intracellular signalling and cytokine production. We also examined ZAP70 amino acids sequences prone to isoaspartyl modification under inflammatory stress and their role in p-Tyr signal transduction, effects on downstream functional domains, and binding to cbl-b.
Results PBMCs from SLE patients and from MRL lupus both have elevated levels of intracellular isoaspartyl modifications and hyperproliferative T cell responses. We identified 4 specific sites of isoAsp modification (Figure 1), two within the I-B functional domain of ZAP70, including the c-Cbl, Vav and Lck binding domain. IsoAsp modified ZAP70 reduces c-Cbl binding, upregulates TCR and T cell hyperplasia. Enzymatic repair of intracellular isoAsp modification corrects T cell hyperproliferative defects that are characteristic of murine and human SLE.
Conclusions This study has examined mechanisms of altered T cell autoimmunity in SLE. In particular, abnormal T cell hyperproliferation was found to be a result of isoaspartyl modification at 4 specific sites within ZAP70. Only a small number of PTMs are known to arise in the context of inflammation. Our study suggests that SLE is characterised by an inability to control or repair excessive production of PTMs due to inflammation, leading to altered cell biologic functions, specifically T cell hyperproliferation. Physiologic repair of intracellular isoAsp modifications reversed abnormal proliferative T cell responses and may provide one therapeutic pathway for intervention.
Posttranslational Protein Modifications in ZAP70 in SLE
Acknowledgements This study is supported by NIH AI48120.