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Raman-Based in Situ Monitoring of Changes in Molecular Signatures during Mitochondrially Mediated Apoptosis.

ABSTRACT: Obtaining molecular information from inside cells is an important topic to understand the outcome of molecular interactions between potential drug molecules and biomolecules inside cells. To envision this goal, we investigated the surface-enhanced Raman scattering-based single-cell spectroscopic method to monitor changes in intracellular molecular signatures during mitochondrially mediated apoptosis in real time. Triphenylphosphine-modified gold nanoparticles were localized successfully to the mitochondria and greatly enhanced to obtain the intrinsic Raman scattering spectrum of mitochondria and cytochrome c in the live cell. Photothermally induced apoptosis showed a moderate decrease in the disulfide bond and a sharp increase in ?-sheet structures depending on the input-laser power, along with morphological changes. However, chemical drug induced-apoptosis showed more subtle changes in the disulfide bond, as well as changes in Raman peaks corresponding to cytochrome c, and the appearance of a new peak at 1420 cm^-1, which enabled us to study the molecular interactions within the mitochondria in real time from a single cell, following treatment with a novel pyruvate dehydrogenase kinase inhibitor.

SUBMITTER: Shin HJ

PROVIDER: S-EPMC6648662 | biostudies-literature | 2019 May

REPOSITORIES: biostudies-literature

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Raman-Based in Situ Monitoring of Changes in Molecular Signatures during Mitochondrially Mediated Apoptosis.

Shin Hyeon Jeong HJ Lee Ji Hye JH Kim Yong Duk YD Shin Injae I Sim Taebo T Lim Dong-Kwon DK

ACS omega 20190506 5

Obtaining molecular information from inside cells is an important topic to understand the outcome of molecular interactions between potential drug molecules and biomolecules inside cells. To envision this goal, we investigated the surface-enhanced Raman scattering-based single-cell spectroscopic method to monitor changes in intracellular molecular signatures during mitochondrially mediated apoptosis in real time. Triphenylphosphine-modified gold nanoparticles were localized successfully to the m ...[more]

PMID: 31459907

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Project description:Targeting the tumor suppressor p53 to the mitochondria triggers a rapid apoptotic response as efficiently as transcription-dependent p53. (1, 2) p53 forms a complex with the antiapoptotic Bcl-XL, which leads to Bak and Bax oligomerization resulting in apoptosis via mitochondrial outer membrane permeabilization. (3, 4) Although p53 performs its main role in the mitochondrial outer membrane, it also interacts with different proteins in the mitochondrial inner membrane and matrix. (5, 6) To further investigate mitochondrial activity of p53, EGFP-p53 was fused to different mitochondrial targeting signals (MTSs) directing it to the mitochondrial outer membrane ("XL-MTS" from Bcl-XL; "TOM-MTS" from TOM20), the inner membrane ("CCO-MTS" from cytochrome c oxidase), or matrix ("OTC-MTS" from ornithine transcarbamylase). Fluorescence microscopy and a p53 reporter dual luciferase assay demonstrated that fusing MTSs to p53 increased mitochondrial localization and nuclear exclusion depending on which MTS was used. To examine if the MTSs initiate mitochondrial damage, we fused each individual MTS to EGFP (a nontoxic protein) as negative controls. We performed caspase-9, TUNEL, annexin-V, and 7-AAD apoptosis assays on T47D breast cancer cells transfected with mitochondrial constructs. Except for EGFP-XL, apoptotic potential was observed in all MTS-EGFP-p53 and MTS-EGFP constructs. In addition, EGFP-p53-XL showed the greatest significant increase in programmed cell death compared to its nontoxic MTS control (EGFP-XL). The apoptotic mechanism for each construct was further investigated using pifithrin-? (an inhibitor of p53 transcriptional activity), pifithrin-? (a small molecule that reduces binding of p53 to Bcl-2 and Bcl-XL), and overexpressing the antiapoptotic Bcl-XL. Unlike the MTSs from TOM, CCO, and OTC, which showed different apoptotic mechanisms, we conclude that p53 fused to the MTS from Bcl-XL performs its apoptotic potential exclusively through the p53/Bcl-XL specific pathway.

Dataset Information

Raman-Based in Situ Monitoring of Changes in Molecular Signatures during Mitochondrially Mediated Apoptosis.

Publications

Raman-Based in Situ Monitoring of Changes in Molecular Signatures during Mitochondrially Mediated Apoptosis.

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