Anti-allergic therapy targeting to a mast cell-specific signaling protein

MIST是一种肥大细胞特异性信号蛋白，在结构上与SLP-76和BLNK/BASH/SLP-65造血细胞特异性接头蛋白相关。我们利用blnk缺失的鸡DT40 B细胞系统，构建了MIST在免疫受体信号传导中的功能分析系统，并筛选了MIST抑制剂以开发抗过敏药物。在这个重建系统中，MIST可以部分恢复blnk缺陷细胞中的B细胞抗原受体（BCR）信号，这需要磷酸化两个n端酪氨酸残基。LAT与MIST共表达完全恢复了BCR信号通路，并且免除了MIST中两种酪氨酸对BCR信号通路的要求。然而，与LAT合作，BCR信号的完全重建仍然需要一些其他酪氨酸，以及MIST中的SH2结构域和两个富含脯氨酸的区域。尽管MIST的c端脯氨酸丰富区域负责与LAT的相互作用以及在富含糖脂的微域中定位，但它对于LAT辅助的MAP激酶激活的完全恢复是必不可少的。另一方面，富含脯氨酸的n端区域是PLCg SH3结构域的结合位点，对BCR信号传导至关重要。我们还证明了肥大细胞系中表达的两个主要的与Mist相关的磷酸化蛋白是SLAP-130和SKAP55，它们是src家族蛋白酪氨酸激酶（PTKs）的SH2结构域的接头，Mist通过其SH2结构域直接与SLAP-130相关，并且SLAP-130与SKAP-55的合作是将Mist募集到Lyn的必要条件。此外，MIST被优先招募到Fyn而不是Lyn，这是由SLAP-130和SKAP55与Fyn- sh2结构域的高亲和力结合而不是Lyn- sh2结构域调节的。综上所述，本研究获得的这些关于MIST在免疫受体信号传导中的功能的信息，为未来利用该MIST重组的blnk缺陷DT40系统开发抗过敏药物奠定了基础

MIST is a mast cell-specific signaling protein structurally related to SLP-76 and BLNK/BASH/SLP-65 hematopoietic cell-specific adaptor proteins. By using the BLNK-deficient DT40 chicken B cell system, we constructed the system for analysing the function of MIST in immunoreceptor signaling and also for screening the MIST inhibitors to develop anti-allergic drugs. In this reconstitution system, MIST can partially restore the B cell antigen receptor (BCR) signaling in the BLNK-deficient cells, which requires phosphorylation of the two N-terminal tyrosine residues. Co-expression of LAT with MIST fully restored the BCR signaling, and dispenses with the requirement of the two tyrosines in MIST for BCR signaling. However, some other tyrosine( s ), as well as the SH2 domain and the two proline-rich regions in MIST are still required for full reconstitution of the BCR signaling, in cooperation with LAT. The C-terminal proline-rich region of MIST is dispensable for the LAT-aided full restoration … More of MAP kinase activation, although it is responsible for the interaction with LAT and for the localization in glycolipidenriched microdomains. On the other hand, the N-terminal proline-rich region, which is a binding site of the SH3 domain of PLCg, is essential for BCR signaling. We also demonstrated that two major MIST-associated phosphoproteins expressed in mast cell lines are SLAP-130 and SKAP55, adaptors known to with the SH2 domain of Src-family protein tyrosine kinases (PTKs), Mist directly associated with SLAP-130 via its SH2 domain, and collaboration of SLAP-130 with SKAP-55 was required for the recruitment of MIST to Lyn. Farthermore, MIST was preferentially recruited to Fyn rather than Lyn, which is regulated by higher affinity binding of SLAP-130 and SKAP55 with the Fyn-SH2 domain than the Lyn-SH2 domain. Taken together, these informations on the function of MIST in immunoreceptor signaling obtained in the present study have established the basis on the future development of antiallergic drugs using this MIST-reconstituted BLNK-deficient DT40 system Less