Targeted degradation of extracellular mitochondrial aspartyl-tRNA synthetase modulates immune responses.

The severity of bacterial pneumonia can be worsened by impaired innate immunity resulting in ineffective pathogen clearance. We describe a mitochondrial protein, aspartyl-tRNA synthetase (DARS2), which is released in circulation during bacterial pneumonia in humans and displays intrinsic innate immune properties and cellular repair properties. DARS2 interacts with a bacterial-induced ubiquitin E3 ligase subunit, FBXO24, which targets the synthetase for ubiquitylation and degradation, a process that is inhibited by DARS2 acetylation. During experimental pneumonia, Fbxo24 knockout mice exhibit elevated DARS2 levels with an increase in pulmonary cellular and cytokine levels. In silico modeling identified an FBXO24 inhibitory compound with immunostimulatory properties which extended DARS2 lifespan in cells. Here, we show a unique biological role for an extracellular, mitochondrially derived enzyme and its molecular control by the ubiquitin apparatus, which may serve as a mechanistic platform to enhance protective host immunity through small molecule discovery.

MicroID2: A Novel Biotin Ligase Enables Rapid Proximity-Dependent Proteomics.

Identifying protein-protein and other proximal interactions is central to dissecting signaling and regulatory processes in cells. BioID is a proximity-dependent biotinylation method that uses an "abortive" biotin ligase to detect proximal interactions in cells in a highly reproducible manner. Recent advancements in proximity-dependent biotinylation tools have improved efficiency and timing of labeling, allowing for measurement of interactions on a cellular timescale. However, issues of size, stability, and background labeling of these constructs persist. Here we modified the structure of BioID2, derived from Aquifex aeolicus BirA, to create a smaller, highly active, biotin ligase that we named MicroID2. Truncation of the C terrminus of BioID2 and addition of mutations to alleviate blockage of biotin/ATP binding at the active site of BioID2 resulted in a smaller and highly active construct with lower background labeling. Several additional point mutations improved the function of our modified MicroID2 construct compared with BioID2 and other biotin ligases, including TurboID and miniTurbo. MicroID2 is the smallest biotin ligase reported so far (180 amino acids [AAs] for MicroID2 versus 257 AAs for miniTurbo and 338 AAs for TurboID), yet it demonstrates only slightly less labeling activity than TurboID and outperforms miniTurbo. MicroID2 also had lower background labeling than TurboID. For experiments where precise temporal control of labeling is essential, we in addition developed a MicroID2 mutant, termed lbMicroID2 (low background MicroID2), that has lower labeling efficiency but significantly reduced biotin scavenging compared with BioID2. Finally, we demonstrate utility of MicroID2 in mass spectrometry experiments by localizing MicroID2 constructs to subcellular organelles and measuring proximal interactions.

Exposure to neighborhood green space and sleep: evidence from the Survey of the Health of Wisconsin.

INTRODUCTION: Adequate sleep duration and quality are protective against many adverse health outcomes. Many individual-level predictors of poor sleep have been examined, but few studies have examined neighborhood-level influences. Despite known associations between neighborhood green space and sleep influencing factors (eg, physical activity, mental health), few studies have examined green space and sleep's relationship. Furthermore, little work has examined the relationship between the magnitude and type of neighborhood sounds and sleep. STUDY METHODS: We analyzed data from the Survey of the Health of Wisconsin database (n = 2712) for 2008-2013, a representative sample of Wisconsin residents ages 21-74. Outcomes included weekday and weekend sleep duration and self-rated sleep quality. Primary predictors were the proportion tree canopy (National Land Cover Database) and mean decibel levels of outdoor sound (US National Park Service) at the census block group level. Survey regression analysis was used to examine statistical associations, controlling for individual and neighborhood-level covariates. RESULTS: Models suggest a significant relationship (P < .05) between weekday sleep duration and green space, and between weekend/day sleep duration and human-made and total neighborhood sound. Increased percent tree canopy in a census block group was associated with lower odds of short weekday sleep (<6 hours) (OR 0.76 [0.58-0.98]). Increased human-made and total mean decibel levels were associated with increased instances of short weekend and weekday sleep (OR 1.05 [1.01-1.08] and 1.03 [1.01-1.06] respectively). CONCLUSIONS: Neighborhood tree canopy and sound levels may influence sleep duration and are potential targets for neighborhood-level interventions to improve sleep.