Abstract
Mitochondria are involved in many cellular processes; mitochondrial dysfunctions have been associated with apoptosis, aging, and a number of pathological conditions, including osteoarthritis (OA). Mitochondrial proteins are attractive targets for the study of metabolism of the chondrocyte, the unique cell type present in mature cartilage, and its role in tissue degradation. Using a proteomics approach based on two-dimensional DIGE and MALDI-TOF/TOF mass spectrometric identification of mitochondria- enriched protein fractions from human articular chondrocytes, we analyzed mitochondrial protein changes that are characteristic of OA chondrocytes. A total of 73 protein forms were unambiguously identified as significantly altered in OA; 23 of them have been previously described as mitochondrial. An extensive statistical and cluster analysis of the data revealed a mitochondrial protein profile characteristic for OA. This pattern includes alterations in energy production, maintenance of mitochondrial membrane integrity, and free radical detoxification. Real time PCR, Western blot, and immunohistofluorescence assays confirmed a significant decrease of the major mitochondrial antioxidant protein manganese-superoxide dismutase (SOD2) in the superficial layer of OA cartilage. As possible outputs for this antioxidant deficiency, we found an increase of intracellular reactive oxygen species generation in OA chondrocytes and also verified an OA-dependent increase in the mitochondrial tumor necrosis factor-α receptor-associated protein 1 (TRAP1), a chaperone with a reported reactive oxygen species antagonist role. Our results describe the differences between the mitochondrial protein profiles of normal and OA chondrocytes, demonstrating that mitochondrial dysregulation occurs in cartilage cells during OA and highlighting redox imbalance as a key factor in OA pathogenesis.
Highlights
Mitochondria are involved in many cellular processes; mitochondrial dysfunctions have been associated with apoptosis, aging, and a number of pathological conditions, including osteoarthritis (OA)
Transcription and translation take place in mitochondria, which actively import proteins and metabolites from the cytosol, influence programmed cell death, and respond to cellular signals such as oxidative stress [1]. In addition to their central role in energy metabolism, mitochondria are involved in many cellular processes; mitochondrial dysfunctions have been associated with apoptosis, aging, and a number of pathological conditions, including Parkinson disease, diabetes mellitus, Alzheimer disease, and OA1 [1,2,3]
Other reports implicate decreased mitochondrial bioenergy reserve as a pathogenic factor in degenerative cartilage disease (9 –11). These findings suggest that mitochondrial proteins would be an attractive target for study of the metabolism of chondrocytes and the role they play in cartilage degradation
Summary
Chemicals, and Antibodies—Culture media and FCS were from Invitrogen. Culture flasks and plates were purchased from Costar (Cambridge, MA). The DIA data sets from each individual gel were collectively analyzed using the biological variation analysis (BVA) module, which allows intergel matching and calculation of average abundance for each protein spot among the six gels of our study. Unsupervised principal component analysis (PCA), hierarchical clustering (HC), and k-means clustering analyses were performed using the DeCyder extended data analysis module on the group of spots identified as significantly changed These multivariate analyses clustered the individual Cy3- and Cy5-labeled samples based on collective comparison of expression patterns from the set of proteins. Database Search—The monoisotopic peptide mass fingerprinting data obtained from MS and the amino acid sequence tag obtained from each peptide fragmentation in MS/MS analyses were used to search for protein candidates using Mascot version 1.9 from Matrix Science.
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