Mitochondrial dysfunction remodels one-carbon metabolism in human cells.

Mitochondrial dysfunction is associated with a spectrum of human disorders, ranging from rare, inborn errors of metabolism to common, age-associated diseases such as neurodegeneration. How these lesions give rise to diverse pathology is not well understood, partly because their proximal consequences have not been well-studied in mammalian cells. Here we provide two lines of evidence that mitochondrial respiratory chain dysfunction leads to alterations in one-carbon metabolism pathways. First, using hypothesis-generating metabolic, proteomic, and transcriptional profiling, followed by confirmatory experiments, we report that mitochondrial DNA depletion leads to an ATF4-mediated increase in serine biosynthesis and transsulfuration. Second, we show that lesioning the respiratory chain impairs mitochondrial production of formate from serine, and that in some cells, respiratory chain inhibition leads to growth defects upon serine withdrawal that are rescuable with purine or formate supplementation. Our work underscores the connection between the respiratory chain and one-carbon metabolism with implications for understanding mitochondrial pathogenesis.

Variability in G-protein-coupled signaling studied with microfluidic devices.

Different cells, even those that are genetically identical, can respond differently to identical stimuli, but the precise source of this variability remains obscure. To study this problem, we built a microfluidic experimental system which can track responses of individual cells across multiple stimulations. We used this system to determine that amplitude variation in G-protein-activated calcium release in RAW264.7 macrophages is generally extrinsic, i.e., they arise from long-lived variations between cells and not from stochastic activation of signaling components. In the case of responses linked to P2Y family purine receptors, we estimate that approximately one-third of the observed variability in calcium release is receptor-specific. We further demonstrate that the signaling apparatus downstream of P2Y6 receptor activation is moderately saturable. These observations will be useful in constructing and constraining single-cell models of G protein-coupled calcium dynamics.

Suppression of LPS-induced TNF-alpha production in macrophages by cAMP is mediated by PKA-AKAP95-p105.

The activation of macrophages through Toll-like receptor (TLR) pathways leads to the production of a broad array of cytokines and mediators that coordinate the immune response. The inflammatory potential of this response can be reduced by compounds, such as prostaglandin E(2), that induce the production of cyclic adenosine monophosphate (cAMP). Through experiments with cAMP analogs and multigene RNA interference (RNAi), we showed that key anti-inflammatory effects of cAMP were mediated specifically by cAMP-dependent protein kinase (PKA). Selective inhibitors of PKA anchoring, time-lapse microscopy, and RNAi screening suggested that differential mechanisms of PKA action existed. We showed a specific role for A kinase-anchoring protein 95 in suppressing the expression of the gene encoding tumor necrosis factor-alpha, which involved phosphorylation of p105 (also known as Nfkb1) by PKA at a site adjacent to the region targeted by inhibitor of nuclear factor kappaB kinases. These data suggest that crosstalk between the TLR4 and cAMP pathways in macrophages can be coordinated through PKA-dependent scaffolds that localize specific pools of the kinase to distinct substrates.