ABSTRACT
Ubiquinone (UQ) is a lipophilic electron carrier that functions in the respiratory and photosynthetic electron transfer chains of proteobacteria and eukaryotes. Bacterial UQ biosynthesis is well studied in the gammaproteobacterium Escherichia coli, in which most bacterial UQ-biosynthetic enzymes have been identified. However, these enzymes are not always conserved among UQ-containing bacteria. In particular, the alphaproteobacterial UQ biosynthesis pathways contain many uncharacterized steps with unknown features. In this work, we identified in the alphaproteobacterium Rhodobacter capsulatus a new decarboxylative hydroxylase and named it UbiN. Remarkably, the UbiN sequence is more similar to a salicylate hydroxylase than the conventional flavin-containing UQ-biosynthetic monooxygenases. Under aerobic conditions, R. capsulatus ΔubiN mutant cells accumulate 3-decaprenylphenol, which is a UQ-biosynthetic intermediate. In addition, 3-decaprenyl-4-hydroxybenzoic acid, which is the substrate of UQ-biosynthetic decarboxylase UbiD, also accumulates in ΔubiN cells under aerobic conditions. Considering that the R. capsulatus ΔubiD-X double mutant strain (UbiX produces a prenylated FMN required for UbiD) grows as a wild-type strain under aerobic conditions, these results indicate that UbiN catalyzes the aerobic decarboxylative hydroxylation of 3-decaprenyl-4-hydroxybenzoic acid. This is the first example of the involvement of decarboxylative hydroxylation in ubiquinone biosynthesis. This finding suggests that the C1 hydroxylation reaction is, at least in R. capsulatus, the first step among the three hydroxylation steps involved in UQ biosynthesis. Although the C5 hydroxylation reaction is often considered to be the first hydroxylation step in bacterial UQ biosynthesis, it appears that the R. capsulatus pathway is more similar to that found in mammalians.
Subject(s)
Rhodobacter capsulatus , Animals , Rhodobacter capsulatus/genetics , Ubiquinone , Mixed Function Oxygenases/genetics , Escherichia coli/genetics , MammalsABSTRACT
BACKGROUND: Spinal cord injury (SCI) in young adults leads to severe sensorimotor disabilities as well as slowing of growth. Systemic pro-inflammatory cytokines are associated with growth failure and muscle wasting. Here we investigated whether intravenous (IV) delivery of small extracellular vesicles (sEVs) derived from human mesenchymal stem/stromal cells (MSC) has therapeutic effects on body growth and motor recovery and can modulate inflammatory cytokines following severe SCI in young adult rats. METHODS: Contusional SCI rats were randomized into three different treatment groups (human and rat MSC-sEVs and a PBS group) on day 7 post-SCI. Functional motor recovery and body growth were assessed weekly until day 70 post-SCI. Trafficking of sEVs after IV infusions in vivo, the uptake of sEVs in vitro, macrophage phenotype at the lesion and cytokine levels at the lesion, liver and systemic circulation were also evaluated. RESULTS: An IV delivery of both human and rat MSC-sEVs improved functional motor recovery after SCI and restored normal body growth in young adult SCI rats, indicating a broad therapeutic benefit of MSC-sEVs and a lack of species specificity for these effects. Human MSC-sEVs were selectively taken up by M2 macrophages in vivo and in vitro, consistent with our previous observations of rat MSC-sEV uptake. Furthermore, the infusion of human or rat MSC-sEVs resulted in an increase in the proportion of M2 macrophages and a decrease in the production of the pro-inflammatory cytokines tumour necrosis factor-alpha (TNF-α) and interleukin (IL)-6 at the injury site, as well as a reduction in systemic serum levels of TNF-α and IL-6 and an increase in growth hormone receptors and IGF-1 levels in the liver. CONCLUSIONS: Both human and rat MSC-sEVs promote the recovery of body growth and motor function after SCI in young adult rats possibly via the cytokine modulation of growth-related hormonal pathways. Thus, MSC-sEVs affect both metabolic and neurological deficits in SCI.
