The activity of mitochondria induces, as a byproduct, a variety of post-translational modifications in associated proteins, which have functional downstream consequences for processes such as apoptosis, autophagy, and plasticity; e.g., reactive oxygen species (ROS), which induce N-formyl-kynurenine from oxidized tryptophans in certain mitochondrial proteins which are localized in close spatial proximity to their source. This type of fast molecular changes has profound influence on cell death and survival with implications in a number of pathologies. The quantitative and differential analysis of bovine heart mitochondria by four 2D-PAGE methods, including 2D-PAGE with high-resolution IEF as first dimension, revealed that due to limited resolution, those methods employing blue native-, tricine-urea-, and 16-BAC-PAGE as the first dimension are less applicable for the differential quantitative analysis of redundant protein spots which might give insight into post-translational modifications that are relevant in age- and stress-related changes. Moreover, 2D-PAGE with high resolution IEF was able to resolve a surprisingly large number of membrane proteins from mitochondrial preparations. For aconitase-2, an enzyme playing an important role in mitochondrial aging, a more thorough molecular analysis of all separable isoforms was performed, leading to the identification of two particular N-formylkynurenine modifications. Next to protein redundancy, native protein-protein interactions, with the potential of relating certain post-translational modification patterns to distinct oligomeric states, e.g., oxidative phosphorylation super complexes, might provide novel and (patho-) physiologically relevant information. Among proteins identified, 14 new proteins (GenBank entries), previously not associated with mitochondria, were found.

The results provide strong evidence for a significant impact of synovial-derived MMPs on cartilage destruction in OA. In this context, fibroblasts present in the synovial fluid appeared to play an outstanding role.

Monoclonal antibodies that recognize HSP70 family members from evolutionarily divergent organisms were used to identify both constitutively-expressed and stress-inducible HSP70 proteins in the green alga Chlamydomonas. These monoclonal antibodies also cross-reacted with a 70 kDa flagellar protein that comigrated with the constitutively-expressed HSP70 isoform(s) present in the cell body; this is the first identification of a molecular chaperone within the eukaryotic flagellum. Fractionation experiments demonstrated that much of the flagellar HSP70 was bound to the ‘9+2’ microtubule axoneme. Incubation of isolated axonemes in ATP, but not AMP or AMP-PNP, caused significant release of the previously bound HSP70 as is characteristic of complexed HSP70s. Immunofluorescent localization in whole flagella showed that flagellar HSP70 was concentrated at the distal ends of flagella, sites of axonemal assembly in vivo. Extraction of axonemes under ionic conditions known to cause the release of capping structures that link the distal ends of the axonemal microtubules to the flagellar membrane also caused the release of axonemal-bound HSP70. Taken together, these results suggest a model in which an HSP70 chaperone may assist in targeting tubulin and other unassembled axonemal components to the flagellar tip where the chaperone may also participate in the assembly of the ‘9+2’ flagellar axoneme.

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