AB0135 PROTEIN CONFORMATIONAL CHANGES AND FUNCTIONAL ALTERATIONS IN DERMAL FIBROBLASTS FROM PATIENTS WITH SYSTEMIC SCLEROSIS

Abstract

BackgroundDespite intensive studies and clinical trials, there is still no available precise diagnostic tool and treatment of the patients affected by systemic sclerosis (SSc). Proteopathies are diseases characterized by the production of aberrant conformers of certain proteins that lead to a disturbance of their cellular functions and disease. It is believed that the structure of a protein is principally responsible for its function. By identifying altered protein structures on a proteome-wide scale, there is a possibility to detect protein functional changes.ObjectivesTo screen for changes in the protein conformation and to correlate with cell function in SSc dermal fibroblasts compared to fibroblasts from healthy donors (HC) using a novel approach of limited proteolysis followed by functional assays.MethodsDiffuse cutaneous SSc and HC dermal fibroblasts were used for recently established Limited Proteolysis-coupled Mass-Spectrometry (Lip-MS)[1] to examine protein structural alterations in a proteome-wide scale. A change in conformation was defined as having a foldchange greater than 1 or smaller than -1 and a significant p-value of -log10 > 1.3 (≤0.05). Further, the responsive signalling targets in these cells, including the NF-κB-dependent pathway and energy metabolism, were evaluated. Fibroblasts were stimulated with inflammatory cytokines, highly relevant in SSc, including TNFα, IL-1β, TGF-β, IL-17A, and a combination of IL-17A and TGF-β. To examine the NF-κB activity, cells were transduced with a pseudo-typed HIV-1-based lentiviral vector. The measurements of luciferase signal were analysed. RT-qPCR was used to assess the expression of NF-κB-dependent genes for non-transduced cells. ATP measurements were analysed and presented as the amount of luminescent signal.ResultsLiP-MS analysis detected 53263 common peptides in SSc (n=6) and HC fibroblasts (n=6), of which 41 peptides showed conformational changes in SSc fibroblasts in comparison with HC fibroblasts. The 41 conformationally altered peptides showed significant enrichment in GO pathways for: biological processes (9), molecular function (7) and cellular components (21). SAE1, CTNND1, CDC37, and PPP1R13L were related to the NF-κB-dependent pathways, while ATP5A1, GSTM1, PCK2, ANPEP, GM2A, GNS, CAD, ACOT2 were connected with metabolic processes. There was a tendency of lower levels of mRNA RELA, NFKBIA, MMP1 and TNC expression levels in untreated and stimulated SSc fibroblasts (n=6) compared to HC fibroblasts (n=6). The assessment of the NF-κB activity in untreated SSc fibroblasts (n=9) showed a trend to lower fold change compared to HC fibroblasts (n=9) (0.63 vs 1, SE 0.19, p=0.07). A statistically significant difference between SSc (n=8) and HC (n=8) fibroblasts was found in TGF-β stimulated fibroblasts (0.64 vs 1, SE 0.16, p=0.046). SSc fibroblasts (n=9) showed a lower ATP level compared to HC fibroblasts (n=9) in untreated condition (0.82 vs 1, SE 0.09, p=0.07), after stimulation with IL-1β (0.85 vs 1, SE 0.08, p=0.07) and TGF-β (0.81 vs 1, SE 0.09, p=0.05). Of note, a statistically significant difference between SSc and HC fibroblasts was found in IL-17A stimulated cells (0.82 vs 1, SE 0.07, p=0.02).ConclusionLiP-MS approach allowed for the identification of conformational changes in SSc fibroblast mainly related to signal transduction and metabolic pathways. Confirmatory functional studies showed deregulated NF-κB activity and ATP levels in SSc fibroblasts. Therefore, LiP-MS approach may create a distinctive opportunity to discover new disease biomarkers and functionally transformed pathways. This method may advance therapeutical approaches, where only structurally altered proteins could be specifically targeted, without interfering with non-changed proteins.