Study On The Mechanism Of Pro-apoptotic BH3-only Protein Tbid Making A "Pore" On The Membrane

Programmed cell death is gene dependent,evolutionarily conserved vital process with the aim of selectively removing the needless cells in multicellutar organisms.According to the different mechanisms,programmed cell death can be mainly divided into three categories,including apoptosis,autophagy and necroptosis.In apoptosis,Bcl-2 superfamily members are the key regulators of the intrinsic apoptosis pathway.Their relative activities directly regulate the mitochondrial outer membrane permeabilization(MOMP).The proapoptotic BH3-only protein Bid,can be activated by the cell death stimuli and then translocate to the mitochondrial outer membrane,where it activate and recruit Bax or Bak to assemble on the membrane inducing the pores and MOMP.Recently,some researches show that tBid itself could also induce MOMP and lysosomal membrane permeabilization(LMP).Researchers found that tBid without the help of other proteins is sufficient to induce LMP,and they detected the release of cathepsin B from the lysosome.This phenomenon also appeared in the Bax and Bak double knocked out cells.They proposed that phosphatidic acid(PA)is the key molecule that bridges tBid and the lysosomal-like membrane permeabilization.However,it is still debatable that whether t Bid could induce pores large enough to releace contents in organelles.In order to end this debate,we used giant unilamellar vesicles(GUVs),which were widely applied in the study of protein and membrane interactions mimicking the organelle membranes,to explore the interaction between tBid and membranes at the single vesicle level.We applied the surface induced fluorescence attenuation(SIFA)to explore the molecular mechanism of tBid induced membrane permeabilization and the effect of ionic strength on tBid conformation at the single molecule level.SIFA takes advantages of the fluorescence quenching property of graphene oxide.By simply labelling the site of interest with fluorophores,we converted the information of protein insertion depths in the membrane into relatice fluorescence intensities.This method with high vertical resolution,which could be sub-nanometer,is easy,and only need traditional single molecule fluorescence equipments.Combining with the single particle tracking,we could get the three dimensional movements of the sites of interest.The development of this method is just for the research of protein and membrane interactions at the single molecule level.Combined the results of the single vesicle level dyes release experiments with the ensemble dyes release experiments and single molecule fluorescence research,we suggested that tBid could induce the permeabilization of membranes containing 20 % DOPA mimicking the lysosomal membranes.Further,it could form small “pores” on the membrane.We proposed the mechanism of this is tBid assembly on the membrane at the single molecule level.Using SIFA,we found that upon binding to the membrane,monomeric tBid inserts into the membrane superficially with residues of 80,140 and 181 on the membrane surface and residue 166 inserting into the membrane about 0.6 ± 0.3 nm below the surface.This suggested that H3 bound to the membrane surface and the central hydrophobic helices H6 and H7 inserted shallowly.With tBid assembled on the membrane to form small oligomers,we found residue 80 got into the solution,while residue 166 underwent slow dynamic conformation changes to insert deeper,about 2.0 ± 0.4 nm beneath the membrane.At the same time,residue 140 and 181 were still at the membrane surface.This meant H3 in small oligomers would change its conformation and go to the solution,while H6 and H7 inserted deeper.When the oligomers became larger,residues of 80,166 and 181 all could get up and down in the membrane among three or even four relatively stable states.Moreover,they all could get to the deep lower leaflet of the membrane transiently.This meant many helices changed their conformations seriously.Although tBid has no static transmembrane domain,here we showed t Bid had dynamically transmembrane domains.We proposed this is the mechanism of tBid induced membrane permeabilization.Further,combined our results with the previous researches,we proposed that tBid could induce toroidal pores on the membrane mimicking the lysosomal membrane.We did research about the impact of ionic strength on tBid and membrane interactions.Our results revealed that it affected the conformation of H3 on the membrane,while had little effect on the H6 and H7 of monomeric tBid on the membrane and the amount of the absorbed proteins on the membrane.Given the above,we demonstrated tBid could induce membrane permeabilization at the single vesicle level and suggested the mechanism of it was tBid form oligomers on the membrane.With SIFA,we analyzed the details of the conformation changes of tBid interacting with membranes mimicking the lysosomal membranes and proposed that tBid induce “pores” on the membrane by forming dynamically transmembrane domains.Further,we suggested that tBid could induce toroidal pores on the membrane.We also found the effect of ionic strength on the conformation of tBid on the membrane.