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.
Aziridinomitosane ketones 4 and 24 are accessed by internal acyl anion equivalent-lactam cyclization of 29 in a convergent route. The key aziridinolactam 6 is prepared by tin-lithium exchange via the lithiated aziridine 11.Mitomycin C (1) and FR900482 (2) are known for their anti-cancer activity, 1 and for their remarkable reductive activation pathway via bis-electrophilic aziridinomitosenes that crosslink DNA. 2 It has been hypothesized that these structurally distinct heterocycles not only share key functionality and mode of activation, but that they also have partially overlapping biosynthetic pathways. 3 Early common biosynthetic intermediates have been identified using isotopic labeling studies, 4 but the point where the biosynthetic pathways diverge is still unknown. In principle, the point of divergence can be investigated using pyrrolo [1,2a]indole-derived aziridines such as 3; however, synthetic access to derivatives of 3 is challenging due to the expected facile aromatization to the indole. 5 With the ultimate goal of synthesizing analogues of 3, we have developed an enantiocontrolled route to the more stable 6 9-oxo-pyrrolo[1,2a]indole 4 as described below. The new route also allows access to the ring system of FR900482 (2) by oxidative ring expansion. 7 A convergent synthetic strategy designed to access the tetracyclic ketol 4 is outlined in Scheme 1, based on the coupling of an aromatic subunit 7 with the bicyclic aziridinolactam 6 followed by C(9)-C(9a) bond formation (mitomycin numbering) using intramolecular nucleophilic addition to the lactam carbonyl group of 5. The aziridine stereochemistry of 5 might be expected to control hemiaminal configuration in 4 based on kinetic or thermodynamic factors. Depending on the outcome of the cyclization step, the C(9a) hemiaminal stereochemistry might then be exploited as one of the factors that set configuration at C(9) corresponding to either the mitomycin or the FR900482 series.First, the chiral bicyclic aziridinolactam 6 was targeted (Scheme 2). Although a number of related bicyclic aziridines are known, 8 no bicyclic NH lactam analog of 6 has been reported. We sought to establish convenient access to 6 and to obtain the aziridine stereocenters from the chiral pool, starting with the known chiral aziridinol 8. 9 Conversion into mesylate 9 (MsCl, Et 3 N, 99%) and azide 10 (91%) was followed by reduction and subsequent treatment of the unstable amine with ethyl chloroformate to provide 11 (81%, 2 steps). Carbamate 11 was converted to dianion 12, which was then trapped with iethyl carbonate to afford edved@umich.edu. Supporting Information Available. Experimental procedures and characterization data for new substances. With bicyclic aziridinolactam 6 in hand, conditions were screened for coupling with aryl iodides containing functionality that might eventually be used to form the tetracycle C(9)-C(9a) bond. The best yields of coupling products were obtained using CuI with K 3 PO 4 and the highly active trans-N,N′-dimethyl-1,2-cyclohexanediamine liga...
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...
The syntheses and reactivity of N-TBDPS and N-trityl protected derivatives of an aziridinomitosene corresponding to FK317 are described. New reactivity patterns were observed for these highly sensitive and functionally dense heterocycles under mild nucleophilic conditions approaching the threshold for degradation. Thus, the silyl or trityl protected aziridinomitosene reacted with Cs2CO3/CD3OD to give isomeric products where substitution occurred at C(10) and C(9a) (mitomycin numbering) providing a CD3 ether and a CD3 hemiaminal respectively. These findings show that heterolysis at C(10) is faster than at aziridine C(1), in contrast to the behavior of typical aziridinomitosenes in the mitomycin series. The labile N-TBDPS hemiaminal and the more stable N-trityl hemiaminal resemble the mitomycin K substitution pattern. A reagent consisting of CsF in CF3CH2OH/CH3CN desilylated a simple N-TBDPS aziridine, but caused nucleophilic cleavage at C(1) as well as C(10) without cleavage of the N-TBPDS group in the fully functionalized penultimate aziridinomitosene. The high reactivity of the C(10) carbamate with nucleophiles precludes the use of deprotection methodology that requires N-protonation for fully functionalized aziridinomitosenes in the FK317 series.
A multimodal activity-based probe for targeting acidic organelles was developed to measure subcellular native enzymatic activity in cells by fluorescence 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 cells 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 the number of tagged proteins and an increase in cathepsin activity. The organelle-targeting of activity-based probes holds great promise for the characterization of enzyme activities in the myriad diseases linked to specific subcellular locations, particularly the lysosome.
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