Pyruvate formate-lyase (acetyl-CoA:formate C-acetyltransferase, EC 2.3.1.54) from anaerobic Escherchia coli cells converts pyruvate to acetyl-CoA and formate by a unique homolytic mechanism that involves a free radical harbored in the protein structure. By EPR spectroscopy of selectively 13C-labeled enzyme, the radical (g = 2.0037) has been assigned to carbon-2 of a glycine residue. Estimated hyperfine coupling constants to the central 13C nucleus (Au = 4.9 mT and A, = 0.1 mT) and to 13C nuclei in a and 13 positions agree with literature data for glycine radical models. N-coupling was verified through uniform I'N-labeling. The large IH hyperfine splitting (1.5 mT) dominating the EPR spectrum was asignd to the a proton, which in the enzyme radical is readily solvent-exchangeable. 996The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.

Suppressor of cytokine signaling (SOCS) proteins constitute a class of negative regulators for Janus kinase/ signal transducer and activator of transcription (JAK/ STAT) signaling pathways. These intracellular proteins are induced by cytokine signaling, but they can also be induced by stimulation of Toll-like receptors (TLR). It has even been suggested that SOCS proteins are important negative regulators of TLR signaling. Here we have elucidated the nature of the regulatory role of SOCS in TLR signaling. Induction of SOCS-3 and cytokine-inducible Src homology 2-containing protein (CIS) by TLR stimulation was strictly dependent on MyD88 but showed differing needs in case of SOCS-1. However, induction of SOCS proteins by TLR ligands was independent of type I interferon. In macrophages overexpressing SOCS, we were not able to observe an inhibitory effect of SOCS-1, SOCS-2, SOCS-3, or CIS on prototypical TLR target genes such as tumor necrosis factor-␣. However, we found that TLR-2, TLR-3, TLR-4, and TLR-9 stimulation induced interferon-␤ (IFN-␤), which is able to exert auto-and paracrine signaling, leading to the activation of secondary genes like IP-10. SOCS-1 and, to a lesser extent, SOCS-3 and CIS were able to inhibit this indirect signaling pathway following TLR stimulation, whereas neither MAP kinase nor NFB signaling were affected. However, STAT-1 tyrosine phosphorylation following TLR triggering was severely impaired by SOCS-1 overexpression. Thus, our data suggest that SOCS proteins induced by TLR stimulation limit the extent of TLR signaling by inhibiting type I IFN signaling but not the main NFB pathway.

In the dentate gyrus – a key component of spatial memory circuits – granule cells (GCs) are known to be morphologically diverse and to display heterogeneous activity profiles during behavior. To resolve structure–function relationships, we juxtacellularly recorded and labeled single GCs in freely moving rats. We found that the vast majority of neurons were silent during exploration. Most active GCs displayed a characteristic spike waveform, fired at low rates and showed spatial activity. Primary dendritic parameters were sufficient for classifying neurons as active or silent with high accuracy. Our data thus support a sparse coding scheme in the dentate gyrus and provide a possible link between structural and functional heterogeneity among the GC population.DOI: http://dx.doi.org/10.7554/eLife.20252.001

Neurons coding for head-direction are crucial for spatial navigation. Here we explored the cellular basis of head-direction coding in the rat dorsal presubiculum (PreS). We found that layer2 is composed of two principal cell populations (calbindin-positive and calbindin-negative neurons) which targeted the contralateral PreS and retrosplenial cortex, respectively. Layer3 pyramidal neurons projected to the medial entorhinal cortex (MEC). By juxtacellularly recording PreS neurons in awake rats during passive-rotation, we found that head-direction responses were preferentially contributed by layer3 pyramidal cells, whose long-range axons branched within layer3 of the MEC. In contrast, layer2 neurons displayed distinct spike-shapes, were not modulated by head-direction but rhythmically-entrained by theta-oscillations. Fast-spiking interneurons showed only weak directionality and theta-rhythmicity, but were significantly modulated by angular velocity. Our data thus indicate that PreS neurons differentially contribute to head-direction coding, and point to a cell-type- and layer-specific routing of directional and non-directional information to downstream cortical targets.DOI: http://dx.doi.org/10.7554/eLife.14592.001

Pyruvate formate-lyase (also called formate acetyltransferase; EC 2.3.1.54; PFI .) catalyses the thiolytic cleavage of pyruvate by CoA, yielding acetyl-CoA and formate. This reaction is the key step in the glucose-fermentation route in Escherichziz coli and various other bacteria. Operationally, it resembles the (B-keto)thiolase reaction of the fatty-acid degradation cycle. The mechanism of pyruvate formate-lyase, however, is fundamentally different, since the carbon-carbon bond of its substrate is cleaved homolytically rather than heterolytically. This property emerged with the discovery of a protein-based radical in the active enzyme form [ 11. The unpaired spin has recently been assigned to C-2 of (;lyi" [ 2 ] .The radical is produced by a postribosomal hydrogen-atom abstraction that is catalysed by PFI, activase using adenosylmethionine (AdoMet) and reduced flavodoxin as co-substrates [ 11. A separate reaction that quenches the protein radical in PFI, is catalysed by the multifunctional AdhE protein and is initiated when anaerobic cells are shifted to positive redox potentials [3].Metabolic aspects of PFI, interconversion between inactive (E) and active (Em) forms and the genetic/transcriptional background of the system have already been reviewed 141. This review will focus on enzyme-catalytic structure/function properties.