In Situ Raman Spectroscopic Characterisation Of Mitochondria-Mediated Cell Death And Its Molecular Regulatory Mechanisms

Mitochondria are not only the energy metabolism center of cells,but also the hub of cell survival and death signal transduction.Mitochondria play a crucial role in cell death pathways.An in-depth understanding of the molecular regulatory mechanisms in the mitochondria-mediated cell death pathway has important research implications for the development of pro-cell death anticancer drugs.Relevant studies have shown that the interaction between cytochrome c（Cyt c）released by mitochondria and the endoplasmic reticulum monooxygenase（MMO）system may accelerate apoptosis;on the other hand,ferroptosis inducers may act on the mitochondrial membrane and cause its dysfunction.However,the specific molecular mechanisms are not clear enough.Raman spectroscopy can provide structural information of biomolecules under physiological conditions,and the rapid development of Surface Enhanced Raman Spectroscopy（SERS）and Surface Enhanced Resonance Raman Spectroscopy（SERRS）has made it possible to characterize biomolecules in complex systems in highly sensitive as well as in situ manner.In this thesis,Raman spectroscopy and SERRS spectroscopy were used as the main research methods to explore the molecular mechanism of mitochondria-endoplasmic reticulum interaction in accelerating apoptosis and mitochondria-mediated apoptosis-ferroptosis interaction.The main research contents and research results obtained are as follows:1.In situ SERRS characterization of ROS production by monooxygenasesSilver nanoparticles,as highly active SERS materials,can significantly enhance the Raman signals of Cyt c.However,direct contact between silver and proteins can easily cause protein denaturation.Based on the good biocompatibility of nickel and its specific electron donor properties for the oxidized state Cyt c(Cyt c_（ox）),we synthesized silver core nickel shell（Ag@Ni）nanostructures.The material has both Cyt c electron donor and Raman signal enhancement properties,which can protect the native conformation of Cyt c.The reduced Cyt c(Cyt c_（red）)will undergo a series of structural changes when attacked by ROS,including axial ligand replacement and loss,and the transformation of the six-coordinated low-spin state B1[6c LS]into B2[6c LS]or five-coordinated high-spin state B2[5c HS]and heme degradation,etc.SERRS can reflect these changes,thus establishing a SERRS method for in situ detection of ROS produced by endoplasmic reticulum monooxygenase（MMO）.2.In situ SERRS monitoring of endoplasmic reticulum-mitochondria crosstalk regulating Cyt c releaseCyt c released from mitochondria was selectively detected by SERRS because Cyt c could not receive electrons from nickel again due to ROS-induced Cyt c denaturation,providing a new research method for in situ characterization of mitochondria-endoplasmic reticulum interactions to accelerate cell apoptosis.The interaction between the anticancer drug methyl 3-（4-nitrophenyl）propiolate（NPP）and the MMO system was investigated,and the ability to induce ROS production by MMO was confirmed by the characteristic Raman spectra of NPP.Furthermore,Ag@Ni nanoparticles were introduced into the isolated mitochondria-endoplasmic reticulum system,and SERRS was used to prove that the addition of NPP can accelerate the release of Cyt c in mitochondria,thereby establishing a non-labelled,real-time,in-situ SERRS method for assessing the pro-apoptotic effects of NPP.The feasibility of this method was finally verified in He La cells.3.Cyt c involved in ferroptosis though Fenton reaction and phospholipid peroxidationThe reaction between Cyt c and H₂O₂ was explored.Electron paramagnetic resonance（EPR）spectroscopy and colorimetric experiments have been used to prove that H₂O₂ can undergo a Fenton-like reaction with heme–Fe in Cyt c,which in turn leads to the degradation of heme and the release of iron ions,initiating the Fenton reaction and triggering ferroptosis.Using matrix-assisted laser desorption ionization-time of flight（MALDI-TOF）mass spectrometry,the ROS-independent phospholipid peroxidation induced by Cyt c-phospholipid interaction was revealed.The relationship between phospholipid peroxidation and membrane permeability was further explored,and Raman results showed that lipid peroxidation increased the permeability of phospholipid membranes.There is a specific interaction between Cyt c and cardiolipin（CL）.Regardless of the presence or absence of ROS,the interaction between the two can induce peroxidation of phospholipids,and Cyt c_（red）disturbs the order of the CL alkyl chain more.It is more conducive to improving the permeability of the phospholipid membrane.These research results prove that Cyt c can participate in the ferroptosis process by releasing Fe ions and specifically interacting with CL and inducing phospholipid peroxidation.4.Ferroptosis inducer erastin triggers apoptosisErastin is cell permeable and can induce mitochondrial voltage-dependent anion channel（VDACs）opening and mitochondrial outer membrane（OMM）permeability changes.In situ Raman spectroscopy and molecular dynamics simulation（MD）were used to study the molecular mechanism of erastin affecting the permeability of phospholipid membranes.The relative intensities of the Raman spectral peaks I₂₉₃₁/I₂₈₅₁showed that erastin hardly interfered with the permeability of the DOPC membrane,but had a significant effect on the phospholipid membrane containing CL.Using acetonitrile as a Raman reporter further confirmed that erastin-CL interaction can perturb mitochondrial membrane permeability.At the same time,the molecular structural basis of the interaction between erastin and phospholipids was explored through MD simulation.The results show that erastin interferes with the ordering of phospholipid membranes throughσ-πinteractions with the hydrophobic tails of phospholipids.Finally,by introducing Ag@Ni nanoparticles,in situ SERRS detection of erastin-induced Cyt c release was achieved.These findings demonstrate that erastin can change the mitochondrial membrane permeability and promote the release of Cyt c by interacting with CL,thereby participating in the apoptosis process.5.Monooxygenase is involved in cell death through phospholipid peroxidationMonooxygenase Cytochrome P450 3A4（CYP 3A4）can induce hydroxylation of unsaturated phospholipids.This chapter examines whether MMO can participate in cell death through mitochondrial phospholipid peroxidation.Firstly,using Raman spectroscopy and MALDI-TOF mass spectrometry,it was found that MMO has a certain interference effect on the ordering of DOPC and CL membranes,and peroxide production were also detected,indicating that MMO can directly participate in cell death through the mitochondrial membrane.Further,when Fe ions were present in this system,it was found that Fe ions interacted with ROS generated by MMO to trigger the Fenton reaction and accelerate phospholipid membrane peroxidation.Meanwhile,the change in Raman intensity of acetonitrile molecules further confirmed that CL with high degree of unsaturation is more susceptible to peroxidation by MMO and MMO/Fe³⁺,causing changes in the permeability of the phospholipid membrane.These results prove that the MMO system on the endoplasmic reticulum can induce mitochondrial membrane peroxidation through its own enzymatic activity or induce Fenton reaction,thereby participating in the process of apoptosis or ferroptosis.