Mapping Salivary Proteases in Sjögren's Syndrome Patients Reveals Overexpression of Dipeptidyl Peptidase-4/CD26.

Sjögren's Syndrome (SS) is an autoimmune exocrinopathy characterized by the progressive damage of salivary and lacrimal glands associated with lymphocytic infiltration. Identifying new non-invasive biomarkers for SS diagnosis remains a challenge, and alterations in saliva composition reported in patients turn this fluid into a source of potential biomarkers. Among these, proteases are promising candidates since they are involved in several key physio-pathological processes. This study evaluated differentially expressed proteases in SS individuals' saliva using synthetic fluorogenic substrates, zymography, ELISA, and proteomic approaches. Here we reported, for the first time, increased activity of the serine protease dipeptidyl peptidase-4/CD26 (DPP4/CD26) in pSS saliva, the expression level of which was corroborated by ELISA assay. Gelatin zymograms showed that metalloproteinase proteolytic band profiles differed significantly in intensity between control and SS groups. Focusing on matrix metalloproteinase-9 (MMP9) expression, an increased tendency in pSS saliva (p = 0.0527) was observed compared to the control group. Samples of control, pSS, and sSS were analyzed by mass spectrometry to reveal a general panorama of proteases in saliva. Forty-eight protein groups of proteases were identified, among which were the serine proteases cathepsin G (CTSG), neutrophil elastase (ELANE), myeloblastin (PRTN3), MMP9 and several protease inhibitors. This work paves the way for proteases to be explored in the future as biomarkers, emphasizing DPP4 by its association in several autoimmune and inflammatory diseases. Besides its proteolytic role, DPP4/CD26 acts as a cell surface receptor, signal transduction mediator, adhesion and costimulatory protein involved in T lymphocytes activation.

Prolyl Oligopeptidase From Leishmania infantum: Biochemical Characterization and Involvement in Macrophage Infection.

Leishmania infantum is a flagellated protozoan and one of the main causative agents of visceral leishmaniasis. This disease usually affects the human reticuloendothelial system, can cause death and available therapies may lead to serious side effects. Since it is a neglected tropical disease, the incentives for the development of new drugs are insufficient. It is important to know Leishmania virulence factors that contribute most to the disease in order to develop drugs. In the present work, we have produced L. infantum prolyl oligopeptidase (rPOPLi) in Escherichia coli, and investigated its biochemical properties as well as the effect of POP inhibitors on its enzymatic activity and on the inhibition of the macrophage infection by L. infantum. The optimal activity occurred at pH 7.5 and 37°C in the presence of DTT, the latter increased rPOPLi catalytic efficiency 5-fold on the substrate N-Suc-Gly-Pro-Leu-Gly-Pro-AMC. The enzyme was inhibited by TPCK, TLCK and by two POP specific inhibitors, Z-Pro-prolinal (ZPP, IC50 4.2 nM) and S17092 (IC50 3.5 nM). Besides being a cytoplasmic enzyme, POPLi is also found in punctuate structures within the parasite cytoplasm or associated with the parasite plasma membrane in amastigotes and promastigotes, respectively. Interestingly, S17092 and ZPP prevented parasite invasion in murine macrophages, supporting the involvement of POPLi in the invasive process of L. infantum. These data suggest POPLi as a virulence factor that offers potential as a target for designing new antileishmanial drugs.

The Pharmacopea within Triatomine Salivary Glands.

Triatomines are blood-feeding insects that prey on vertebrate hosts. Their saliva is largely responsible for their feeding success. The triatomine salivary content has been studied over the past decades, revealing multifunctional bioactive proteins targeting the host´s hemostasis and immune system. Recently, sequencing of salivary-gland mRNA libraries revealed increasingly complex and complete transcript databases that have been used to validate the expression of deduced proteins through proteomics. This review provides an insight into the journey of discovery and characterization of novel molecules in triatomine saliva.