New structural insights into the role of TROVE2 complexes in the on-set and pathogenesis of systemic lupus erythematosus determined by a combination of QCM-D and DPI.

The mechanism of self-recognition of the autoantigen TROVE2, a common biomarker in autoimmune diseases, has been studied with a quartz crystal microbalance with dissipation monitoring (QCM-D) and dual polarization interferometry (DPI). The complementarity and remarkable analytical features of both techniques has allowed new insights into the onset of systemic lupus erythematosus (SLE) to be achieved at the molecular level. The in vitro study for SLE patients and healthy subjects suggests that anti-TROVE2 autoantibodies may undergo an antibody bipolar bridging. An epitope-paratope-specific binding initially occurs to activate a hidden Fc receptor in the TROVE2 tertiary structure. This bipolar mechanism may contribute to the pathogenic accumulation of anti-TROVE2 autoantibody immune complex in autoimmune disease. Furthermore, the specific calcium-dependent protein-protein bridges point out at how the TRIM21/TROVE2 association might occur, suggesting that the TROVE2 protein could stimulate the intracellular immune signaling via the TRIM21 PRY-SPRY domain. These findings may help to better understand the origins of the specificity and affinity of TROVE2 interactions, which might play a key role in the SLE pathogenesis. This manuscript gives one of the first practical applications of two novel functions (-df/dD and Δh/molec) for the analysis of the data provided by QCM-D and DPI. In addition, it is the first time that QCM-D has been used for mapping hidden Fc receptors as well as linear epitopes in a protein tertiary structure. Graphical abstract ᅟ.

Mapping molecular binding by means of conformational dynamics measurements.

Protein-protein interactions are key in virtually all biological processes. The study of these interactions and the interfaces that mediate them play a key role in the understanding of biological function. In particular, the observation of protein-protein interactions in their dynamic environment is technically difficult. Here two surface analysis techniques, dual polarization interferometry and quartz crystal microbalance with dissipation monitoring, were paired for real-time mapping of the conformational dynamics of protein-protein interactions. Our approach monitors this dynamics in real time and in situ, which is a great advancement within technological platforms for drug discovery. Results agree with the experimental observations of the interaction between the TRIM21α protein and circulating autoantibodies via a bridging bipolar mechanism. This work provides a new chip-based method to monitor conformational dynamics of protein-protein interactions, which is amenable to miniaturized high-throughput determination.

Label-free piezoelectric biosensor for prognosis and diagnosis of Systemic Lupus Erythematosus.

An autoantigen piezoelectric sensor to quantify specific circulating autoantibodies in human serum is developed. The sensor consisted on a quartz crystal microbalance with dissipation monitoring (QCM-D) where TRIM21 and TROVE2 autoantigens were covalently immobilized, allowing the selective determination of autoantibodies for diagnosis and prognosis of Systemic Lupus Erythematosus (SLE). The sensitivity of the biosensor, measured as IC50 value, was 1.51U/mL and 0.32U/mL, for anti-TRIM21 and anti-TROVE2 circulating autoantibodies, respectively. The sensor is also able to establish a structural interaction fingerprint pattern or profile of circulating autoantibodies, what allows scoring accurately SLE patients. Furthermore, a statistical association of global disease activity with TRIM21-TROVE2 interaction was found (n=130 lupic patient samples, p-value=0.0413). The performances of the biosensor were compared with standard ELISA and multiplex DVD-array high-throughput screening assays, corroborating the viability of piezoelectric biosensor as a cost-effective in vitro assay for the early detection, monitoring or treatment of rare diseases.