Direct detection of the myosin super-relaxed state and interacting-heads motif in solution.

The interacting-heads motif (IHM) is a structure of myosin that has been proposed to modulate cardiac output by occluding myosin molecules from undergoing the force-generating cycle. It is hypothesized to be the structural basis for the super-relaxed state (SRX), a low-ATPase kinetic state thought to be cardioprotective. The goal of the present study was to test this hypothesis by determining directly and quantitatively the fractions of myosin in the IHM and SRX under the same conditions in solution. To detect the structural IHM, we used time-resolved fluorescence resonance energy transfer to quantitate two distinct populations. One population was observed at a center distance of 2.0 nm, whereas the other was not detectable by fluorescence resonance energy transfer, implying a distance greater than 4 nm. We confirmed the IHM assignment to the 2.0-nm population by applying the same cross-linking protocol used previously to image the IHM by electron microscopy. Under the same conditions, we also measured the fraction of myosin in the SRX using stopped-flow kinetics. Our results show that the populations of SRX and IHM myosin were similar, unless treated with mavacamten, a drug that recently completed phase III clinical trials to treat hypertrophic cardiomyopathy and is proposed to act by stabilizing both the SRX and IHM. However, we found that mavacamten had a much greater effect on the SRX (55% increase) than on the IHM (4% increase). We conclude that the IHM structure is sufficient but not necessary to produce the SRX kinetic state.

Continuous treatment with IL-15 exhausts human NK cells via a metabolic defect.

NK cell-based immunotherapies have been gaining traction in the clinic for treatment of cancer. IL-15 is currently being used in number of clinical trials to improve NK cell expansion and function. The objective of this study is to evaluate the effect of repetitive IL-15 exposure on NK cells. An in vitro model in which human NK cells are continuously (on on on) or intermittently (on off on) treated with IL-15 was used to explore this question. After treatment, cells were evaluated for proliferation, survival, cell cycle gene expression, function, and metabolic processes. Our data indicate that continuous treatment of NK cells with IL-15 resulted in decreased viability and a cell cycle arrest gene expression pattern. This was associated with diminished signaling, decreased function both in vitro and in vivo, and reduced tumor control. NK cells continuously treated with IL-15 also displayed a reduced mitochondrial respiration profile when compared with NK cells treated intermittently with IL-15. This profile was characterized by a decrease in the spare respiratory capacity that was dependent on fatty acid oxidation (FAO). Limiting the strength of IL-15 signaling via utilization of an mTOR inhibitor rescued NK cell functionality in the group continuously treated with IL-15. The findings presented here show that human NK cells continuously treated with IL-15 undergo a process consistent with exhaustion that is accompanied by a reduction in FAO. These findings should inform IL-15-dosing strategies in NK cell cancer immunotherapeutic settings.