A systematic assessment of mycobacterial F1 -ATPase subunit ε's role in latent ATPase hydrolysis.

In contrast to most bacteria, the mycobacterial F1 FO -ATP synthase (α3 :ß3 :γ:δ:ε:a:b:b':c9 ) does not perform ATP hydrolysis-driven proton translocation. Although subunits α, γ and ε of the catalytic F1 -ATPase component α3 :ß3 :γ:ε have all been implicated in the suppression of the enzyme's ATPase activity, the mechanism remains poorly defined. Here, we brought the central stalk subunit ε into focus by generating the recombinant Mycobacterium smegmatis F1 -ATPase (MsF1 -ATPase), whose 3D low-resolution structure is presented, and its ε-free form MsF1 αßγ, which showed an eightfold ATP hydrolysis increase and provided a defined system to systematically study the segments of mycobacterial ε's suppression of ATPase activity. Deletion of four amino acids at ε's N terminus, mutant MsF1 αßγεΔ2-5 , revealed similar ATP hydrolysis as MsF1 αßγ. Together with biochemical and NMR solution studies of a single, double, triple and quadruple N-terminal ε-mutants, the importance of the first N-terminal residues of mycobacterial ε in structure stability and latency is described. Engineering ε's C-terminal mutant MsF1 αßγεΔ121 and MsF1 αßγεΔ103-121 with deletion of the C-terminal residue D121 and the two C-terminal ɑ-helices, respectively, revealed the requirement of the very C terminus for communication with the catalytic α3 ß3 -headpiece and its function in ATP hydrolysis inhibition. Finally, we applied the tools developed during the study for an in silico screen to identify a novel subunit ε-targeting F-ATP synthase inhibitor.

Capsinoids activate brown adipose tissue (BAT) with increased energy expenditure associated with subthreshold 18-fluorine fluorodeoxyglucose uptake in BAT-positive humans confirmed by positron emission tomography scan.

Background: Capsinoids are reported to increase energy expenditure (EE) via brown adipose tissue (BAT) stimulation. However, imaging of BAT activation by capsinoids remains limited. Because BAT activation is a potential therapeutic strategy for obesity and related metabolic disorders, we sought to prove that capsinoid-induced BAT activation can be visualized by 18-fluorine fluorodeoxyglucose (18F-FDG) positron emission tomography (PET). Objective: We compared capsinoids and cold exposure on BAT activation and whole-body EE. Design: Twenty healthy participants (8 men, 12 women) with a mean age of 26 y (range: 21-35 y) and a body mass index (kg/m2) of 21.7 (range: 18.5-26.0) underwent 18F-FDG PET and whole-body calorimetry after ingestion of 12 mg capsinoids or ≤2 h of cold exposure (∼14.5°C) in a crossover design. Mean standardized uptake values (SUVs) of the region of interest and BAT volumes were calculated. Blood metabolites were measured before and 2 h after each treatment. Results: All of the participants showed negligible 18F-FDG uptake post-capsinoid ingestion. Upon cold exposure, 12 participants showed avid 18F-FDG uptake into supraclavicular and lateral neck adipose tissues (BAT-positive group), whereas the remaining 8 participants (BAT-negative group) showed undetectable uptake. Capsinoids and cold exposure increased EE, although cold induced a 2-fold increase in whole-body EE and higher fat oxidation, insulin sensitivity, and HDL cholesterol compared with capsinoids. Conclusions: Capsinoids only increased EE in BAT-positive participants, which suggests that BAT mediates EE evoked by capsinoids. This implies that capsinoids stimulate BAT to a lesser degree than cold exposure as evidenced by 18F-FDG uptake below the presently accepted SUV thresholds defining BAT activation. This trial was registered at www.clinicaltrials.gov as NCT02964442.