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.

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.

Aspergillus fumigatus is the primary pathogen causing the devastating pulmonary disease Invasive Aspergillosis in immunocompromised individuals. There is high genomic synteny between A. fumigatus and closely related rarely pathogenic Neosartorya fischeri and Aspergillus clavatus genomes. We applied activity-based protein profiling to compare unique or overexpressed activitybased probe-reactive proteins of all three fungi over time in minimal media growth and in response to human serum. We found 360 probe-reactive proteins exclusive to A. fumigatus, including known virulence associated proteins, and 13 proteins associated with stress response exclusive to A. fumigatus culture in serum. Though the fungi are highly orthologous, A. fumigatus has a significantly greater number of ABP-reactive proteins across varied biological process. Only 50% of expected orthologs of measured A. fumigatus reactive proteins were observed in N. fischeri and A. clavatus. Activity-based protein profiling identified a number of processes that were induced by human serum in A. fumigatus relative to N. fischeri and A. clavatus. These included actin organization and assembly, transport, and fatty acid, cell membrane, and cell wall synthesis. Additionally, signaling proteins regulating vegetative growth, conidiation, and cell wall integrity, required for appropriate cellular response to external stimuli, had higher activity-based probe-protein reaction over time in A. fumigatus and N. fisheri, but not in A. clavatus. Together, we show that measured proteins and physiological processes identified solely or significantly over-represented in A. fumigatus reveal a unique adaptive response to human protein not found in closely related, but rarely pathogenic aspergilli. These unique activity-based probe-protein responses to culture condition may reveal how A. fumigatus initiates pulmonary invasion leading to Invasive Aspergillosis. Invasive aspergillosis (IA) 1 is a devastating infection caused by the ubiquitous saprophytic filamentous fungus Aspergillus fumigatus (Af) (1). Af is an opportunistic pathogen with no true virulence factors. Its pathogenicity is often attributed to its thermotolerance, response to oxidative stress, ability to grow in hypoxic or iron limiting environments, and its ability to use a variety of carbon and nitrogen sources as nutrients, such as proteins derived from the human host (2). A thorough understanding of biological processes or factors that facilitate pathogenic Af infection compared with other microbial infections is needed to assist treatment and diagnosis of IA.Af protein activity regulation and function, attenuated by environmental response and adaptation, is critical for opportunistic infection and development of IA (3). Activity-based protein profiling (ABPP), coupled to mass spectrometry (MS), is a powerful chemical biology approach for directly identifying a subset of proteins (4). ABPP employs activity-based probes (ABPs) to covalently label and enrich functional families of proteins, thereby reducing th...