Gingival proteomics reveals the role of TGF beta and YAP/TAZ signaling in Raine syndrome fibrosis.

Raine syndrome (RNS) is a rare autosomal recessive osteosclerotic dysplasia. RNS is caused by loss-of-function disease-causative variants of the FAM20C gene that encodes a kinase that phosphorylates most of the secreted proteins found in the body fluids and extracellular matrix. The most common RNS clinical features are generalized osteosclerosis, facial dysmorphism, intracerebral calcifications and respiratory defects. In non-lethal RNS forms, oral traits include a well-studied hypoplastic amelogenesis imperfecta (AI) and a much less characterized gingival phenotype. We used immunomorphological, biochemical, and siRNA approaches to analyze gingival tissues and primary cultures of gingival fibroblasts of two unrelated, previously reported RNS patients. We showed that fibrosis, pathological gingival calcifications and increased expression of various profibrotic and pro-osteogenic proteins such as POSTN, SPARC and VIM were common findings. Proteomic analysis of differentially expressed proteins demonstrated that proteins involved in extracellular matrix (ECM) regulation and related to the TGFß/SMAD signaling pathway were increased. Functional analyses confirmed the upregulation of TGFß/SMAD signaling and subsequently uncovered the involvement of two closely related transcription cofactors important in fibrogenesis, Yes-associated protein (YAP) and transcriptional coactivator with PDZ-binding motif (TAZ). Knocking down of FAM20C confirmed the TGFß-YAP/TAZ interplay indicating that a profibrotic loop enabled gingival fibrosis in RNS patients. In summary, our in vivo and in vitro data provide a detailed description of the RNS gingival phenotype. They show that gingival fibrosis and calcifications are associated with, and most likely caused by excessed ECM production and disorganization. They furthermore uncover the contribution of increased TGFß-YAP/TAZ signaling in the pathogenesis of the gingival fibrosis.

Validation in the ESPOIR cohort of vitamin K-dependent protein S (PROS) as a potential biomarker capable of predicting response to the methotrexate/etanercept combination.

BACKGROUND: To validate the ability of PROS (vitamin K-dependent protein S) and CO7 (complement component C7) to predict response to the methotrexate (MTX)/etanercept (ETA) combination in rheumatoid arthritis (RA) patients who received this therapeutic combination in a well-documented cohort. METHOD: From the ESPOIR cohort, RA patients having received the MTX/ETA or MTX/adalimumab (ADA) combination as a first-line biologic treatment were included. Serum concentrations of PROS and CO7 were measured by ELISA prior to the initiation of ETA or ADA, at a time where the disease was active (DAS28 ESR > 3.2). The clinical efficacy (response/non-response) of both combinations has been evaluated after at least 6 months of treatment, according to the EULAR response criteria with some modifications. RESULTS: Thirty-two were treated by MTX/ETA; the numbers of responders and non-responders were 24 and 8, respectively. Thirty-three patients received the MTX/ADA combination; 27 and 5 patients were respectively responders and non-responders. While there were no differences for demographic, clinical, biological, and X-rays data, as well as for CO7, serum levels of PROS tended to be significantly higher in responders to the MTX/ETA combination (p = 0.08) while no difference was observed in the group receiving MTX/ADA. For PROS, the best concentration threshold to differentiate both groups was calculated at 40 µg/ml using ROC curve. The theranostic performances of PROS appeared better for the ETA/MTX combination. When considering the response to this combination, analysis of pooled data from ESPOIR and SATRAPE (initially used to validate PROS and CO7 as potential theranostic biomarkers) cohorts led to a higher theranostic value of PROS that became significant (p = 0.009). CONCLUSION: PROS might be one candidate of a combination of biomarkers capable of predicting the response to MTX/ETA combination in RA patients refractory to MTX. TRIAL REGISTRATION: ClinicalTrials.gov identifiers: NCT03666091 and NCT00234234 .

Physiological and proteomic analyses of Tunisian local grapevine (Vitis vinifera) cultivar Razegui in response to drought stress.

Drought is one of the major environmental constraints threatening viticulture worldwide. Therefore, it is critical to reveal the molecular mechanisms underlying grapevine (Vitis vinifera L.) drought stress tolerance useful to select new species with higher tolerance/resilience potentials. Drought-tolerant Tunisian local grapevine cultivar Razegui was exposed to water deficit for 16days. Subsequent proteomic analysis revealed 49 differentially accumulated proteins in leaves harvested on the drought-stressed vines. These proteins were mainly involved in photosynthesis, stress defence, energy and carbohydrate metabolism, protein synthesis/turnover and amino acid metabolism. Physiological analysis revealed that reduction of photosynthesis under drought stress was attributed to the downregulation of the light-dependent reactions, Calvin cycle and key enzymes of the photorespiration pathway. The accumulation of proteins involved in energy and carbohydrate metabolism indicate enhanced need of energy during active stress acclimation. Accumulation of protein amino acids seems to play a protective role under drought stress due to their osmoprotectant and ROS scavenging potential. Reduced protein synthesis and turnover help plants preserving energy to fight drought stress. Proteins related to stress defence might scavenge ROS and transmit the ROS signal as an oxidative signal transducer in drought-stress signalling. All of these original results represent valuable information towards improving drought tolerance of grapevine and promoting sustainable viticulture under climate change conditions.

Pathogenesis of Enamel-Renal Syndrome Associated Gingival Fibromatosis: A Proteomic Approach.

The enamel renal syndrome (ERS) is a rare disorder featured by amelogenesis imperfecta, gingival fibromatosis and nephrocalcinosis. ERS is caused by bi-allelic mutations in the secretory pathway pseudokinase FAM20A. How mutations in FAM20A may modify the gingival connective tissue homeostasis and cause fibromatosis is currently unknown. We here analyzed conditioned media of gingival fibroblasts (GFs) obtained from four unrelated ERS patients carrying distinct mutations and control subjects. Secretomic analysis identified 109 dysregulated proteins whose abundance had increased (69 proteins) or decreased (40 proteins) at least 1.5-fold compared to control GFs. Proteins over-represented were mainly involved in extracellular matrix organization, collagen fibril assembly, and biomineralization whereas those under-represented were extracellular matrix-associated proteins. More specifically, transforming growth factor-beta 2, a member of the TGFß family involved in both mineralization and fibrosis was strongly increased in samples from GFs of ERS patients and so were various known targets of the TGFß signaling pathway including Collagens, Matrix metallopeptidase 2 and Fibronectin. For the over-expressed proteins quantitative RT-PCR analysis showed increased transcript levels, suggesting increased synthesis and this was further confirmed at the tissue level. Additional immunohistochemical and western blot analyses showed activation and nuclear localization of the classical TGFß effector phospho-Smad3 in both ERS gingival tissue and ERS GFs. Exposure of the mutant cells to TGFB1 further upregulated the expression of TGFß targets suggesting that this pathway could be a central player in the pathogenesis of the ERS gingival fibromatosis. In conclusion our data strongly suggest that TGFß -induced modifications of the extracellular matrix contribute to the pathogenesis of ERS. To our knowledge this is the first proteomic-based analysis of FAM20A-associated modifications.

Anti-Carbamylated Fibrinogen Antibodies Might Be Associated With a Specific Rheumatoid Phenotype and Include a Subset Recognizing In Vivo Epitopes of Its γ Chain One of Which Is Not Cross Reactive With Anti-Citrullinated Protein Antibodies.

To identify the targets recognized by anti-carbamylated protein antibodies (anti-CarP) in patients with early Rheumatoid Arthritis (RA), to study the cross-reactivity between anti-CarP and anti-citrullinated protein antibodies (ACPA) and to evaluate their prognostic value. 331 patients (184 RA and 147 other rheumatisms) from the Very Early Arthritis (VErA) French cohort were analyzed. We performed mass spectrometry analysis of RA sera displaying anti-CarP activity and epitope mapping of the carbamylated fibrinogen γ chain to identify immunodominant peptides. The specificity of these targets was studied using competition assays with the major antigens recognized by ACPA. The prognostic value of anti-carbamylated fibrinogen IgG antibodies (ACa-Fib IgG) was compared to that of anti-cyclic citrullinated peptide antibodies (anti-CCP) and anti-CarP using an in-house ELISA. Besides the α chain, the γ chain of fibrinogen, particularly one immunodominant epitope that has a specific reactivity, was identified as a circulating carbamylated target in sera. The prevalence of ACa-Fib was 37% at baseline and 10.9% for anti-CCP-negative RA. In anti-CCP-negative patients, ACa-Fib positivity was associated with a more inflammatory and erosive disease at baseline but not with rapid radiological progression, which remains strongly related to anti-CCP antibodies. Fibrinogen seems to be one of the antigens recognized in vivo by the anti-CarP response, particularly 2 epitopes of the γ chain, one of which is not cross reactive with ACPA. This specificity might be associated with a distinct clinical phenotype since ACa-Fib IgG were shown to be linked to systemic inflammation in very early RA but not to rapid radiological progression.

3-Benzoylquinoxalinone as a photoaffinity labelling derivative with fluorogenic properties allowing reaction monitoring under "no-wash" conditions.

Described herein is a quinoxalinone-based photoaffinity probe with caged fluorescence properties. Upon visible blue LED irradiation (λmax 450 nm), this photo-crosslinker is able to covalently capture proteins with concomitant fluorescence labelling. This process enables monitoring applications under "no wash" conditions.

Investigation of tetrazine reactivity towards C-nucleophiles: pyrazolone-based modification of biomolecules.

Chemoselective, biocompatible ligation reactions are the key components for efficient and modular access to biomolecular scaffolds. Tetrazine ligation leads to the formation of a mixture of isomers, which makes reaction monitoring, purification and characterization of conjugates difficult. We report herein a modified tetrazine ligation strategy based on the use of a pyrazolone coupling partner, which provides a single molecule conjugate.

Soluble alpha-enolase activates monocytes by CD14-dependent TLR4 signalling pathway and exhibits a dual function.

Rheumatoid arthritis (RA) is the most common form of chronic inflammatory rheumatism. Identifying auto-antigens targeted by RA auto-antibodies is of major interest. Alpha-enolase (ENO1) is considered to be a pivotal auto-antigen in early RA but its pathophysiologic role remains unknown. The main objective of this study was to investigate the in vitro effects of soluble ENO1 on peripheral blood mononuclear cells (PBMC) from healthy donors and RA patients in order to determine the potential pathogenic role of ENO1. ELISA, transcriptomic analysis, experiments of receptor inhibition and flow cytometry analysis were performed to determine the effect, the target cell population and the receptor of ENO1. We showed that ENO1 has the ability to induce early production of pro-inflammatory cytokines and chemokines with delayed production of IL-10 and to activate the innate immune system. We demonstrated that ENO1 binds mainly to monocytes and activates the CD14-dependent TLR4 pathway both in healthy subjects and in RA patients. Our results establish for the first time that ENO1 is able to activate in vitro the CD14-dependent TLR4 pathway on monocytes involving a dual mechanism firstly pro-inflammatory and secondly anti-inflammatory. These results contribute to elucidating the role of this auto-antigen in the pathophysiologic mechanisms of RA.

Dysregulation of RasGRP1 in rheumatoid arthritis and modulation of RasGRP3 as a biomarker of TNFα inhibitors.

BACKGROUND: B and T cells play a key role in rheumatoid arthritis (RA) pathophysiology. RasGRP1 and RasGRP3 are involved in T and B cell receptors signaling, and belong to gene combination able to predict infliximab responsiveness, leading to the question of RasGRP1 and RasGRP3 involvement in RA. METHODS: RasGRP1 and RasGRP3 expression levels were measured by qRT-PCR and/or western-blot in peripheral blood mononuclear cells (PBMCs), in T and B cells from untreated RA patients and in RA patients treated by TNFα inhibitors. T and B cells from healthy controls (HC) were cultured with TNFα, and TNFα receptors neutralizing antibodies to highlight the TNFα effects on RasGRP1 and RasGRP3 pathways. MAPK pathways and apoptosis were respectively analyzed using the Proteome Profiler arrays and flow cytometry. RESULTS: In PBMCs from RA patients, gene expression levels of RasGRP1 were invariant while RasGRP3 was downregulated under TNFα inhibitors and upregulated under TNFα. In T cells from RA patients, RasGRP1 was decreased and its gene expression level was correlated with disease activity. In T cells from HC, TNFα stimulation increased RasGRP1 gene expression level while it reduced RasGRP1 protein expression level. Bryostatin-1 experiments have confirmed that the TNFα effect observed on T cells proliferation was due to the decrease of RasGRP1 expression. Besides, RasGRP3 expression level increased in PBMCs from RA patients under TNFα and in B cells from HC leading us to conclude that RasGRP3 in B cells was modulated by TNFα. CONCLUSION: This study demonstrates RasGRP1 dysregulation in RA patients while RasGRP3 is characterized as a biomarker linked to TNFα inhibitors. After binding to TNFR1, TNFα reduced RasGRP1 protein expression resulting in inhibition of T cell activation. TRIAL REGISTRATION: Clinicaltrials.gov NCT00234234 , registered 04 November 2008; NCT00767325 , registered 05 October 2005.

Prophylactic Injection of Recombinant Alpha-Enolase Reduces Arthritis Severity in the Collagen-Induced Arthritis Mice Model.

OBJECTIVE: To evaluate the ability of the glycolytic enzyme alpha-enolase (ENO1) or its immunodominant peptide (pEP1) to reduce the severity of CIA in DBA/1 mice when injected in a prophylactic way. METHODS: Mice were treated with mouse ENO1 or pEP1 one day prior to collagen II immunization. Clinical assessment was evaluated using 4 parameters (global and articular scores, ankle thickness and weight). Titers of serum anti-ENO1, anti-cyclic citrullinated peptides (anti-CCP) and anti-CII (total IgG and IgG1/IgG2a isotypes) antibodies were measured by ELISA at different time-points. Disease activity was assessed by histological analysis of both anterior and hind paws at the end of experimentation. RESULTS: Prophylactic injection of 100 µg of ENO1 reduced severity of CIA. Serum levels of anti-CII antibodies were reduced in ENO1-treated mice. Concordantly, ENO1-treated mice joints presented less severe histological signs of arthritis. ENO1 did not induce a shift toward a Th2 response since IgG1/IgG2a ratio of anti-CII antibodies remained unchanged and IL-4 serum levels were similar to those measured in the control group. CONCLUSIONS: Pre-immunization with ENO1 or its immunodominant peptide pEP1 reduces CIA severity at the clinical, immunological and histological levels. Effects of pEP1 were less pronounced. This immunomodulatory effect is associated with a reduction in anti-CII antibodies production but is not due to a Th1/Th2 shift.