Reyes Luis Fernando, Nobre Thatyane M, Pavinatto Felippe J, Zaniquelli Maria E D, Caseli Luciano, Oliveira Osvaldo N, Araújo Ana Paula U
Centro de Biotecnologia Molecular Estrutural, Instituto de Física de São Carlos, Universidade de São Paulo, PO Box 369, 13560-970 São Carlos, SP, Brazil.
Biochim Biophys Acta. 2012 Jan;1818(1):82-9. doi: 10.1016/j.bbamem.2011.10.002. Epub 2011 Oct 12.
Pulchellin is a Ribosome Inactivating Protein containing an A-chain (PAC), whose toxic activity requires crossing the endoplasmic reticulum (ER) membrane. In this paper, we investigate the interaction between recombinant PAC (rPAC) and Langmuir monolayers of dipalmitoyl phosphatidyl glycerol (DPPG), which served as membrane model. Three catalytically active, truncated PACs with increasing deletion of the C-terminal region, possessing 244, 239 and 236 residues (rPAC(244), rPAC(239) and rPAC(236)), were studied. rPAC had the strongest interaction with the DPPG monolayer, inducing a large expansion in its surface pressure-area isotherm. The affinity to DPPG decreased with increased deletion of the C-terminal region. When the C-terminal region was deleted completely (rPAC(236)), the interaction was recovered, probably because other hydrophobic regions were exposed to the membrane. Using Polarization Modulated-Infrared Reflection Absorption Spectroscopy (PM-IRRAS) we observed that at a bare air/water interface rPAC comprised mainly α-helix structures, the C-terminal region had unordered structures when interacting with DPPG. For rPAC(236) the α-helices were preserved even in the presence of DPPG. These results confirm the importance of the C-terminal region for PAC-ER membrane interaction. The partial unfolding only with preserved C-terminal appears a key step for the protein to reach the cytosol and develop its toxic activity.
普契林是一种核糖体失活蛋白，含有A链（PAC），其毒性活性需要穿过内质网（ER）膜。在本文中，我们研究了重组PAC（rPAC）与作为膜模型的二棕榈酰磷脂酰甘油（DPPG）的朗缪尔单层之间的相互作用。研究了三种具有催化活性、C末端区域缺失逐渐增加的截短PAC，分别含有244、239和236个残基（rPAC(244)、rPAC(239)和rPAC(236)）。rPAC与DPPG单层的相互作用最强，在其表面压力-面积等温线上引起大幅扩展。随着C末端区域缺失的增加，对DPPG的亲和力降低。当C末端区域完全缺失时（rPAC(236)），相互作用恢复，可能是因为其他疏水区域暴露于膜上。使用偏振调制红外反射吸收光谱（PM-IRRAS），我们观察到在裸露的空气/水界面，rPAC主要由α-螺旋结构组成，C末端区域与DPPG相互作用时具有无序结构。对于rPAC(236)，即使存在DPPG，α-螺旋也得以保留。这些结果证实了C末端区域对于PAC-ER膜相互作用的重要性。仅在保留C末端的情况下部分解折叠似乎是蛋白质到达细胞质并发挥其毒性活性的关键步骤。
