The mitochondrial transcription factor A (mtTFA) is central to assembly and initiation of the mitochondrial transcription complex. Human mtTFA (h-mtTFA) is a dual high mobility group box (HMGB) protein that binds site-specifically to the mitochondrial genome and demarcates the promoters for recruitment of h-mtTFB1, h-mtTFB2 and the mitochondrial RNA polymerase. The stoichiometry of h-mtTFA was found to be a monomer in the absence of DNA, whereas it formed a dimer in the complex with the light strand promoter (LSP) DNA. Each of the HMG boxes and the C-terminal tail were evaluated for their ability to bind to the LSP DNA. Removal of the C-terminal tail only slightly decreased nonsequence specific DNA binding, and box A, but not box B, was capable of binding to the LSP DNA. The X-ray crystal structure of h-mtTFA box B, at 1.35 Å resolution, revealed the features of a noncanonical HMG box. Interactions of box B with other regions of h-mtTFA were observed. Together, these results provide an explanation for the unusual DNA-binding properties of box B and suggest possible roles for this domain in transcription complex assembly.

Resident tissue macrophages are activated by the fungal pathogen Candida albicans to release eicosanoids, which are important modulators of inflammation and immune responses. Our objective was to identify the macrophage receptors engaged by C. albicans that mediate activation of group IVA cytosolic phospholipase A2 (cPLA2α), a regulatory enzyme that releases arachidonic acid (AA) for production of prostaglandins and leukotrienes. A comparison of peritoneal macrophages from wild type and knock-out mice demonstrates that the β-glucan receptor Dectin-1 and MyD88 regulate early release of AA and eicosanoids in response to C. albicans. However, cyclooxygenase 2 (COX2) expression and later phase eicosanoid production are defective in MyD88−/− but not Dectin-1−/− macrophages. Furthermore, C. albicans-stimulated activation of MAPK and phosphorylation of cPLA2α on Ser-505 are regulated by MyD88 and not Dectin-1. In contrast, Dectin-1 mediates MAPK activation, cPLA2α phosphorylation, and COX2 expression in response to particulate β-glucan suggesting that other receptors engaged by C. albicans preferentially mediate these responses. Results also implicate the mannan-binding receptor Dectin-2 in regulating cPLA2α. C. albicans-stimulated MAPK activation and AA release are blocked by d-mannose and Dectin-2-specific antibody, and overexpression of Dectin-2 in RAW264.7 macrophages enhances C. albicans-stimulated MAPK activation, AA release, and COX2 expression. In addition, calcium mobilization is enhanced in RAW264.7 macrophages overexpressing Dectin-1 or -2. The results demonstrate that C. albicans engages both β-glucan and mannan-binding receptors on macrophages that act with MyD88 to regulate the activation of cPLA2α and eicosanoid production.

Cytosolic phospholipase A 2 α (cPLA 2 α, Group IVA phospholipase A 2 ) is a central mediator of arachidonate release from cellular phospholipids for the biosynthesis of eicosanoids. cPLA 2 α translocates to intracellular membranes including the Golgi in response to a rise in intracellular calcium level. The enzyme's calcium-dependent phospholipid-binding C2 domain provides the targeting specificity for cPLA 2 α translocation to the Golgi. However, other features of cPLA 2 α regulation are incompletely understood such as the role of phosphorylation of serine residues in the catalytic domain and the function of basic residues in the cPLA 2 α C2 and catalytic domains that are proposed to interact with anionic phospholipids in the membrane to which cPLA 2 α is targeted. Increasing evidence strongly suggests that cPLA 2 α plays a role in regulating Golgi structure, tubule formation and intra-Golgi transport. For example, recent data suggests that cPLA 2 α regulates the transport of tight junction and adherens junction proteins through the Golgi to cell-cell contacts in confluent endothelial cells. However, there are now examples where data based on knockdown using siRNA or pharmacological inhibition of enzymatic activity of cPLA 2 α affects fundamental cellular processes yet these phenotypes are not observed in cells from cPLA 2 α deficient mice. These results suggest that in some cases there may be compensation for the lack of cPLA 2 α. Thus, there is continued need for studies employing highly specific cPLA 2 α antagonists in addition to genetic deletion of cPLA 2 α in mice.

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