High-fat diet (HFD) induced obesity and concomitant development of insulin resistance (IR) and type 2 diabetes mellitus have been linked to mitochondrial dysfunction. However, it is not clear whether mitochondrial dysfunction is a direct effect of a HFD, or if mitochondrial function is reduced with increased HFD duration. We hypothesized that the function of mitochondrial oxidative and lipid metabolism functions in skeletal muscle mitochondria for HFD mice are similar, or elevated, relative to standard diet (SD) mice; thereby, IR is neither cause nor consequence of mitochondrial dysfunction. We applied a chemical probe approach to identify functionally reactive ATPases and nucleotide-binding proteins in mitochondria isolated from skeletal muscle of C57Bl/6J mice fed HFD or SD chow for 2-, 8-, or 16-weeks; feeding time points known to induce IR. A total of 293 probe-labeled proteins were identified by mass spectrometry-based proteomics, of which 54 differed in abundance between HFD and SD mice. We found proteins associated with the TCA cycle, oxidative phosphorylation (OXPHOS), and lipid metabolism were altered in function when comparing SD to HFD fed mice at 2-weeks, however by 16-weeks HFD mice had TCA cycle, β-oxidation, and respiratory chain function at levels similar to or higher than SD mice.

A multimodal acidic organelle targeting activity-based probe was developed to measure subcellular native enzymatic activity of cells by fluorescent microscopy and mass spectrometry. A cathepsin reactive warhead, conjugated to a weakly basic amine and a clickable alkyne, for subsequent appendage of a fluorophore or biotin reporter tag, accumulated in lysosomes as observed by Structured Illumination Microscopy (SIM) in J774 mouse macrophage cells. Analysis of in vivo labeled J774 by mass spectrometry showed that the probe was very selective for Cathepsins B and Z, two lysosomal cysteine proteases. Analysis of starvation-induced autophagy, a catabolic pathway involving lysosomes, showed a large increase in tagged protein number and an increase in cathepsin activity. Organelle targeting activity-based probes, enabled by fine-tuning of physicochemical features, holds great promise for characterizing enzyme activities in the myriad diseases implicated to subcellular locales, particularly the lysosome.

Background:A. fumigatus is an opportunistic pathogen responsible for pulmonary invasive aspergillosis. Results: Multiplexed ABPP revealed significant changes in A. fumigatus metabolism and stress response during culture with human serum over time. Conclusion: Changes in functional pathways indicate robust adaptation to environmental change. Significance: A. fumigatus grows under stress by altering metabolism, energy production, and protein biosynthesis, which is relevant for lung colonization.

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