The second-sphere residue T263 is important for the function and catalytic activity of PTP1B via interaction with the WPD-loop.

Protein tyrosine phosphatases have diverse substrate specificities and intrinsic activities that lay the foundations for the fine-tuning of a phosphorylation network to precisely regulate cellular signal transduction. All classical PTPs share common catalytic mechanisms, and the important catalytic residues in the first sphere of their active sites have been well characterized. However, little attention has been paid to the second-sphere residues that are potentially important in defining the intrinsic activity and substrate specificity of PTPs. Here, we find that a conserved second-sphere residue, Thr263, located in the surface Q-loop is important for both the function and activity of PTPs. Using PTP1B as a study model, we found that mutations of Thr263 impaired the negative regulation role of PTP1B in insulin signaling. A detailed mechanistic study utilizing steady-state kinetics, Brønsted analysis and pH dependence in the presence of pNPP or phosphopeptide substrates revealed that Thr263 is required for the stabilization of the leaving group during catalysis. Further crystallographic studies and structural comparison revealed that Thr263 regulates the general acid function through modulation of the WPD-loop by the T263:F182/Y/H interaction pair, which is conserved in 26 out of 32 classical PTPs. In addition, the hydrophobic interaction between Thr263 and Arg1159 of the insulin receptor contributes to the substrate specificity of PTP1B. Taken together, our findings demonstrate the general role of the second-sphere residue Thr263 in PTP catalysis. Our findings suggest that the second sphere residues of PTP active site may play important roles in PTP-mediated function in both normal and diseased states.

[IL-25 derived from epithelial cells has the potential to promote airway remodeling in asthma].

AIM: To explore the role of interleukin-25(IL-25) in the pathogenesis of eosinophilic asthma (EA) and non-eosinophilic asthma (NEA) through detecting its expression in serum, induced sputum and bronchial epithelial mucosa of asthmatic patients. METHODS: Serum and induced sputum were collected from 55 untreated asthmatic patients and 27 healthy control subjects. The asthmatic patients were divided into EA and NEA groups according to sputum eosinophils(EOS) percentage (3% as a dividing point). The level of IL-25 in serum and induced spntum was determined by ELISA; the expression of IL-25 in bronchial epithelium was quantified by immunohistochemistry in biopsied specimens from 10 cases of EA, 10 NEA and 10 controls. Basement membrane thickness as an important index of airway remodeling was detected by HE staining. RESULTS: Compared with healthy control subjects, the lung function was impaired in patients with EA and NEA. ELISA results showed that the levels of IL-25 in the serum and induced sputum of asthmatic patients were significantly higher than those in healthy subjects (P<0.05). But there were no statistic differences between EA and NEA patients (P>0.05). The immunohistochemical results indicated that the expression of IL-25 was higher in asthmatic bronchial epithelium than in control ones. HE staining showed that the basement membrane thickness increased in EA and NEA patients(P<0.05). Correlation analysis showed that the levels of IL-25 in serum and induced sputum were positively correlated with the average thickness of basement membrane in asthmatic patients. CONCLUSION: IL-25 secreted from epithelial cells has the potential to promote airway remodeling in asthma. EOS has nothing to do with the thickness of basement membrane, and it may not be necessary for airway remodeling in asthma.

[Expression of MADD in lung adenocarcinoma tissues and its effects on proliferation and apoptosis of lung adenocarcinoma A549 cells].

AIM: To investigate the expression of MAPK-activating death domain protein (MADD) in lung adenocarcinoma tissues and its effects on proliferation and apoptosis of lung adenocarcinoma A549 cells. METHODS: Immunohistochemistry was used to detect the expression of MADD in lung normal and tumor tissues. The expression of IG20 gene in A549 cells was measured by reverse transcription polymerase chain reaction. A549 cells were transfected with pEYFP-MADD plasmids carrying MADD gene or pNL-SIN-GFP-MID lentiviral vectors used for RNA interference. MADD expression and cell proliferation and apoptosis were determined by Western blot, MTT assay, and flow cytometry. RESULTS: The expression levels of MADD were higher in lung adenocarcinoma and squamous cell carcinoma tissues than that in lung normal tissues, and lung adenocarcinoma tissues expressed more MADD than lung squamous cell carcinoma tissues. The transcript encoding MADD was expressed in A549 cells. The transfection of pEYFP-MADD plasmids could increase MADD expression and cell proliferation of A549 cells, while the A549 cells transfected with pNL-SIN-GFP-MID lentiviral vectors showed significantly decreases in the MADD level and proliferation. It is shown that MADD overexpression could inhibit A549 cell apoptosis, and knock down of MADD could promote apoptosis of them. CONCLUSION: The expression of MADD increases obviously in lung adenocarcinoma, and MADD can promote survival of lung adenocarcinoma cells by inhibiting apoptosis.

Thymic stromal lymphopoietin promotes lung inflammation through activation of dendritic cells.

Asthma is an epithelial disorder in which T helper 2 (Th2)-type inflammation has a prominent role. Recent studies indicated that a cytokine, thymic stromal lymphopoietin (TSLP), is essential for the development of antigen-induced asthma. The authors used ovalbumin (OVA) sensitization and challenge to induce a murine asthmatic model. The model was confirmed by airway hyperresponsiveness, serum levels of total and OVA-specific immunoglobulin (IgE), histological analysis of lung tissues. The authors found that expression of TSLP was significantly increased in both mRNA and protein levels in mice lungs treated with OVA. The expression of CD40, CD80, and CD86 in bronchoalveolar lavage fluid (BALF) was increased in mice with OVA. Tight correlation between TSLP mRNA and interleukin (IL)-4, IL-5, and IL-13 in BALF was identified. Furthermore, treating mice with TSLP-neutralizing antibody reduced the expression of TSLP mRNA of lungs, CD40, CD80, and CD86 on dendritic cells, and IL-4, IL-5, and IL-13 in the OVA group. This study indicates that TSLP is increased in the airway epithelium in mice treated with OVA. In the lung inflammation model, TSLP activates dendritic cells (DCs) via up-regulation of CD40, CD80, and CD86, then induces the differentiation of prime naive CD4(+) T cells to become proinflammatory Th2 cells. Blocking TSLP is capable of inhibiting the production of Th2 cytokines, thus presents a promising strategy for the treatment of asthma.

FIZZ1 plays a crucial role in early stage airway remodeling of OVA-induced asthma.

The aim of this study was to elucidate the role of Found in Inflammatory Zone 1 (FIZZ1, also known as RELM-alpha or resistin-like molecule-alpha) in airway remodeling in asthma. We used a rat model of ovalbumin (OVA) sensitization and challenge to induce lung inflammation and remodeling. Expression of alpha -SMA in the lungs of OVA-treated rats was significantly elevated in the peribronchial regions compared with control saline-treated animals. Expression of FIZZ1 mRNA in alveolar epithelial type II cells (AECII) isolated from OVA-treated animals was higher than in control animals. Forced expression of recombinant FIZZ1 in rat-1 lung fibroblast cell line enhanced production of collagen type I and alpha -SMA compared with control transfected cells. These results suggest that FIZZ1 can induce fibroblasts to express markers of myofibroblast differentiation such as alpha -SMA and collagen type I, which are characteristic of early stages of airway remodeling seen in asthma.