Faculty Bibliography

NQO1 [NAD(P)H quinone oxidoreductase 1; also known as DT-diaphorase] is a cytosolic enzyme that catalyses the two-electron reduction of various quinones including vitamin K. The enzyme may play a role in vitamin K metabolism by reducing vitamin K to vitamin K hydroquinone for utilization in the post-translational gamma-glutamyl carboxylation reactions required by several proteins involved in blood coagulation. The aim of the present study was to assess the contribution of NQO1 to vitamin K reduction and haemostasis in an in vivo model. We examined the contribution of NQO1 to haemostasis by examining survival rates in mice poisoned with the anticoagulant warfarin. Supraphysiological amounts of vitamin K sufficiently reversed the effects of warfarin in both wild-type and NQO1-deficient mice. Additionally, vitamin K reductase activities distinct from VKOR (vitamin K epoxide reductase) and NQO1 were measured in vitro from both wild-type and NQO1-defecient mice. The results of the present study suggest that NQO1 does not play a major role in the production of vitamin K hydroquinone and supports the existence of multiple vitamin K reduction pathways. The properties of a NAD(P)H-dependent vitamin K reductase different from NQO1 are described.

The endocardium forms the inner lining of the heart tube, where it enables blood flow and also interacts with the myocardium during the formation of valves and trabeculae. Although a number of studies have identified regulators in the morphogenesis of the myocardium, relatively little is known about the molecules that control endocardial morphogenesis. Prior work has implicated the bHLH transcription factor Tal1 in endocardial tube formation: in zebrafish embryos lacking Tal1, endocardial cells form a disorganized mass within the ventricle and do not populate the atrium. Through blastomere transplantation, we find that tal1 plays a cell-autonomous role in regulating endocardial extension, suggesting that Tal1 activity influences the behavior of individual endocardial cells. The defects in endocardial behavior in tal1-deficient embryos originate during the earliest steps of endocardial morphogenesis: tal1-deficient endocardial cells fail to generate a cohesive monolayer at the midline and instead pack tightly together into a multi-layered aggregate. Moreover, the tight junction protein ZO-1 is mislocalized in the tal1-deficient endocardium, indicating a defect in intercellular junction formation. In addition, we find that the tal1-deficient endocardium fails to maintain its identity; over time, a progressively increasing number of tal1-deficient endocardial cells initiate myocardial gene expression. However, the onset of defects in intercellular junction formation precedes the onset of ectopic myocardial gene expression in the tal1-deficient endocardium. We therefore propose a model in which Tal1 has distinct roles in regulating the formation of endocardial intercellular junctions and maintaining endocardial identity.

E protein transcription factors and their natural inhibitors, Id proteins, play critical and complex roles during lymphoid development. In this article, we report that partial maintenance of E protein activity during positive selection results in a change in the cell fate determination of developing iNKT cells, with a block in the development of iNKT1 cells and a parallel increase in the iNKT2 and iNKT17 subsets. Because the expression levels of the transcription factors that drive these alternative functional fates (GATA-3, RORgammaT, T-bet, and Runx-3) are not altered, our results suggest that E protein activity controls a novel checkpoint that regulates the number of iNKT precursors that choose each fate.

The unfolded protein response (UPR) is composed by homeostatic signaling pathways that are activated by excessive protein misfolding in the endoplasmic reticulum. Ire1 signaling is an important mediator of the UPR, leading to the activation of the transcription factor Xbp1. Here, we show that Drosophila Ire1 mutant photoreceptors have defects in the delivery of rhodopsin-1 to the rhabdomere and in the secretion of Spacemaker/Eyes Shut into the interrhabdomeral space. However, these defects are not observed in Xbp1 mutant photoreceptors. Ire1 mutant retinas have higher mRNA levels for targets of regulated Ire1-dependent decay (RIDD), including for the fatty acid transport protein (fatp). Importantly, the downregulation of fatp by RNAi rescues the rhodopsin-1 delivery defects observed in Ire1 mutant photoreceptors. Our results show that the role of Ire1 during photoreceptor differentiation is independent of Xbp1 function and demonstrate the physiological relevance of the RIDD mechanism in this specific paradigm.

This paper describes a novel application of information-asymmetric (signalling) games to molecular biology in which utility is determined by the message complexity (rate) in addition to the error in information transfer (distortion). We show using a computational model how it is possible for the agents in one such game to evolve a signalling convention (separating equilibrium) that is suboptimal in terms of information transfer, but is nonetheless stable. In the context of an RNA world merging with a nascent amino acid one, such a game's equilibrium is alluded to by the genetic code, which is nearly optimal in terms of information transfer, but is also near-universal and nearly immutable. Such a framework suggests that cellularity may have emerged to encourage coordination between RNA species and sheds light on other aspects of RNA world biochemistry yet to be fully understood.

We previously reported that PGRN directly bound to TNF receptors (TNFR) in vitro and in chondrocytes (Tang, et al., Science, 2011). Here we report that PGRN also associated with TNFR in splenocytes, and inhibited the binding of TNFalpha to immune cells. Proper folding of PGRN is essential for its binding to TNFR, as DTT treatment abolished its binding to TNFR. In contrast, the binding of PGRN to Sortilin was enhanced by DTT. Protein interaction assays with mutants of the TNFR extracellular domain demonstrated that CRD2 and CRD3 of TNFR are important for the interaction with PGRN, similar to the binding to TNFalpha. Taken together, these findings provide the molecular basis underlying PGRN/TNFR interaction and PGRN-mediated anti-inflammatory activity in various autoimmune diseases and conditions.

AIMS: Cell function requires formation of molecular clusters localized to discrete subdomains. The composition of these interactomes, and their spatial organization, cannot be discerned by conventional microscopy given the resolution constraints imposed by the diffraction limit of light ( approximately 200-300 nm). Our aims were (i) Implement single-molecule imaging and analysis tools to resolve the nano-scale architecture of cardiac myocytes. (ii) Using these tools, to map two molecules classically defined as components 'of the desmosome' and 'of the gap junction', and defined their spatial organization. METHODS AND RESULTS: We built a set-up on a conventional inverted microscope using commercially available optics. Laser illumination, reducing, and oxygen scavenging conditions were used to manipulate the blinking behaviour of individual fluorescent reporters. Movies of blinking fluorophores were reconstructed to generate subdiffraction images at approximately 20 nm resolution. With this method, we characterized clusters of connexin43 (Cx43) and of 'the desmosomal protein' plakophilin-2 (PKP2). In about half of Cx43 clusters, we observed overlay of Cx43 and PKP2 at the Cx43 plaque edge. SiRNA-mediated loss of Ankyrin-G expression yielded larger Cx43 clusters, of less regular shape, and larger Cx43-PKP2 subdomains. The Cx43-PKP2 subdomain was validated by a proximity ligation assay (PLA) and by Monte-Carlo simulations indicating an attraction between PKP2 and Cx43. CONCLUSIONS: (i) Super-resolution fluorescence microscopy, complemented with Monte-Carlo simulations and PLAs, allows the study of the nanoscale organization of an interactome in cardiomyocytes. (ii) PKP2 and Cx43 share a common hub that permits direct physical interaction. Its relevance to excitability, electrical coupling, and arrhythmogenic right ventricular cardiomyopathy, is discussed.