tBid蛋白引发磷脂膜透化过程的研究

Fluorescent investigation on process of tBid inducing membrane permeabilization

Bid蛋白是仅有BH3结构域的Bcl-2家族蛋白,在溶酶体膜透化以及线粒体外膜透化引发的细胞凋亡过程中起着非常重要的调控作用,但是Bid蛋白与生物膜之间的相互作用导致脂膜透化的确切机制尚不十分清楚.本文利用激光扫描共聚焦显微成像技术及基于氧化石墨烯表面诱导荧光衰逝的单分子荧光技术,分别从单囊泡及单分子水平对tBid蛋白与磷脂膜之间的相互作用进行了系统的研究.结果表明,tBid蛋白在膜上聚集后可引起脂膜的透化,且脂膜透化的发生源于聚集体中一些tBid蛋白更深入地插入了脂膜中.

The proapoptotic protein tBid is a member of Bcl-2 family, and it plays an important role in apoptosis by inducing mitochondrial outer membrane permeabilization（MOMP） and lysosomal membrane permeabilization（LMP）.Previous studies have shown that the mechanism of tBid-dependent MOMP and LMP depends on tBid interacting with membranes. Researchers hold different opinions about whether tBid itself could induce MOMP and LMP. Some of the researchers insist that tBid must trigger other proteins like Bax or Bak inserting into the membrane, and assembly of tBid itself could not form pores large enough to release cytochrome c. Some others think that tBid just like Bax, can permeabilize mitochondrial outer membrane releasing cytochrome c and lysosomal membrane with the leakage of lysosomal cathepsin B. Here, we want to know whether the tBid itself can induce membrane permeabilization in our model system at low concentration. We use 3 ways to observe tBid and membranes interactions. They are confocal imaging of GUVs（giant unilamellar vesicles）, traditional single molecular fluorescence assay, and a recently developed approach,single molecular surface-induced fluorescence attenuation（sm-SIFA）. So we can obtain information from single vesicle level and single molecule level. At single vesicle level, we can directly find out whether the GUVs are permeabilized and at the same time the shape of the GUVs is changed. At a single molecule level, we can know the properties of one protein. Especially by using the sm-SIFA, we can obtain the insertion depth of exact residue. Combining the results obtained from different ways under the same conditions, we find that tBid itself can induce the model membrane to permeate, releasing the fluorescent molecules, by oligomerization. What is more, we suggest that the mechanism is that in oligomers some tBids can be inserted deep into the membrane although in oligomers not all the proteins have the same insertion depth. It is indicated that the conformations of tBids in oligomers are diversified. We also prove that the ways we use here are efficient. The GUVs and supported lipid bilayers are indeed tenable model systems. Sm-SIFA has a grand future in the study of protein and membrane interactions.