Lorena Herrera1, Mauricio Mastrogiovanni1, Manuela Pose1, Ari Zeida1, Lucía Piacenza1, Rafael Radi1
Cytochrome c (cyt c) is a mitochondrial protein located in the intermembrane space, where it plays a central role in electron transport within the respiratory chain. In the cytosol, cyt c functions as a pro-apoptotic factor. Under conditions of oxidative stress, cyt c undergoes structural modifications that alter its iron coordination center, potentially redirecting its function toward alternative cellular roles. This conformational change can be specifically recognized by the R1D3 antibody and is recapitulated by oxidation of methionine-80, yielding the MetSO-cyt c proteoform. Previous findings from our research group demonstrated that MetSO-cyt c, when delivered to cells via micropinocytosis, is translocated to the nucleus and does not induce apoptosis.
The objective of this study is to investigate the structural changes associated to MetSO-cyt c and to elucidate the functional implications of this proteoform within the cellular context Specifically we aim to elucidate interaction partners and characterize the transcriptional and proteomic changes.
Distinct proteoforms of cyt c were generated: MetSO-cyt c via chloramine-T oxidation and apo-cyt c via silver sulfate treatment. Structural characterization was conducted using UV-vis spectrophotometry, circular dichroism and mass spectrometry.
In order to study the biological implications of MetSO-cyt c, micropinocytosis assays were performed in HeLa cells. Proteomic analyses of cell lysates were conducted to evaluate protein-level changes. Early time points (4 h) were assessed using native cyt c and MetSO-cyt c, while 24 h time points included MetSO-cyt c and a control condition. Protein extraction was followed by trypsin digestion and triplicate analysis using the ZenoTOF7600 system (SCIEX). Chromatographic separation was performed on a reversed-phase bioZen column (2.6 µm Peptide XB-C18, 150 × 2.1 mm, Phenomenex) using an acetonitrile gradient. Data-dependent acquisition was executed in positive mode with a full scan 400–1000 m/z, followed by MS/MS of the top 20 ions (z ≥ +2) per segment using dynamic background subtraction. Bioinformatic analysis was carried out using PatternLab for Proteomics V5. The present work shows the protein identification and differential expression analysis, which will be validated through additional biological replicates and subsequent biochemical assays.
Ongoing experiments include crosslinking followed by immunoprecipitation using the R1D3 antibody on cell lysates, with subsequent mass spectrometric analysis. Gene expression profiling is also underway via transcriptomic analyses following macropinocytosis of cells treated with MetSO-cyt c, native cyt c, and apo-cyt c.
Collectively, this experimental framework aims to advance our understanding of the alternative conformation of cytochrome c and its biological significance at the cellular level.
Agradecimentos: CEINBIO, PAyS, PEDECIBA Biología, ANII, Facultad de Medicina, Universidad de la República