Identification and characterization of D-AKAP1 as a major adipocyte PKA and PP1 binding protein

Protein kinase A (PKA) plays an important role in the regulation of lipid metabolism in adipocytes. The activity of PKA is known to be modulated by its specific location in the cell, a process mediated by A-kinase anchoring proteins (AKAPs). In order to examine the subcellular localization of PKA in this tissue we performed a search for AKAP proteins in adipocytes. We purified a 120 kDa protein which can bind both the regulatory subunit of PKA as well as the catalytic subunit of protein phosphatase 1 (PP1). This protein was found to be enriched in the lipid droplet fraction of primary adipocytes and was identified as D-AKAP1. This protein may play an important role in the regulation of PKA in adipocytes.     

Use of transgenic mice to study lung morphogenesis and function

Successful transition to air breathing at birth depends on perinatal maturation of the gas exchange surface, resorption of fluid from the air spaces, and synthesis and secretion of pulmonary surfactant. Genetic mutations that alter lung development and/or cellular differentiation in the prenatal period, lung function in the perinatal period, or lung homeostasis in the postnatal period can lead to neonatal lethality or chronic lung disease. Current knowledge of the molecular pathways that regulate key prenatal, perinatal, and postnatal morphogenetic events has been shaped largely by remarkable advances in transgenic technologies. In this review, selected transgenic mouse models are highlighted to illustrate the power of this technology, which in many cases has provided important insights that otherwise could not have been obtained.     

Mössbauer spectroscopy on respiratory complex I: the iron-sulfur cluster ensemble in the NADH-reduced enzyme is partially oxidized

In mitochondria, complex I (NADH:quinone oxidoreductase) couples electron transfer to proton translocation across an energy-transducing membrane. It contains a flavin mononucleotide to oxidize NADH, and an unusually long series of iron-sulfur (FeS) clusters that transfer the electrons to quinone. Understanding electron transfer in complex I requires spectroscopic and structural data to be combined to reveal the properties of individual clusters and of the ensemble. EPR studies on complex I from Bos taurus have established that five clusters (positions 1, 2, 3, 5, and 7 along the seven-cluster chain extending from the flavin) are (at least partially) reduced by NADH. The other three clusters, positions 4 and 6 plus a cluster on the other side of the flavin, are not observed in EPR spectra from the NADH-reduced enzyme: they may remain oxidized, have unusual or coupled spin states, or their EPR signals may be too fast relaxing. Here, we use M?ssbauer spectroscopy on (57)Fe-labeled complex I from the mitochondria of Yarrowia lipolytica to show that the cluster ensemble is only partially reduced in the NADH-reduced enzyme. The three EPR-silent clusters are oxidized, and only the terminal 4Fe cluster (position 7) is fully reduced. Together with the EPR analyses, our results reveal an alternating profile of higher and lower potential clusters between the two active sites in complex I; they are not consistent with the consensus picture of a set of isopotential clusters. The implications for intramolecular electron transfer along the extended chain of cofactors in complex I are discussed.     

Rab5 proteins regulate activation and localization of target of rapamycin complex 1

The mechanistic target of rapamycin (mTOR) complex 1 is regulated by small GTPase activators and localization signals. We examine here the role of the small GTPase Rab5 in the localization and activation of TORC1 in yeast and mammalian cells. Rab5 mutants disrupt mTORC1 activation and localization in mammalian cells, whereas disruption of the Rab5 homolog in yeast, Vps21, leads to decreased TORC1 function. Additionally, regulation of PI(3)P synthesis by Rab5 and Vps21 is essential for TORC1 function in both contexts.     

Directing the biological activities of heparan sulfate oligosaccharides using a chemoenzymatic approach

Heparan sulfate (HS) and heparin are highly sulfated polysaccharides exhibiting essential physiological functions. The sulfation patterns determine the functional selectivity for HS and heparin. Chemical synthesis of HS, especially those larger than a hexasaccharide, remains challenging. Enzymatic synthesis of HS has recently gained momentum. Here we describe the divergent assembly of HS heptasaccharides and nonasaccharides from a common hexasaccharide precursor. The hexasaccharide precursor was synthesized via a chemical method. The subsequent elongation, sulfation and epimerization were completed by glycosyltransferases, HS sulfotransferases and epimerase. Using the synthesized heptasaccharides, we discovered that the iduronic acid is critical for binding to fibroblast growth factor-2. We also designed a synthetic path to prepare a nonasaccharide with an antithrombin-binding affinity of 3?nM. Our method demonstrated the feasibility of combining chemical and enzymatic synthesis to prepare structurally defined HS oligosaccharides with desired biological activities.     

Continued optimization of the MLPCN probe ML071 into highly potent agonists of the hM1 muscarinic acetylcholine receptor

This Letter describes the continued optimization of the MLPCN probe molecule ML071. After introducing numerous cyclic constraints and novel substitutions throughout the parent structure, we produced a number of more highly potent agonists of the M(1) mACh receptor. While many novel agonists demonstrated a promising ability to increase soluble APPα release, further characterization indicated they may be functioning as bitopic agonists. These results and the implications of a bitopic mode of action are presented.     

Configuration of a high-content imaging platform for hit identification and pharmacological assessment of JMJD3 demethylase enzyme inhibitors

The biological complexity associated with the regulation of histone demethylases makes it desirable to configure a cellular mechanistic assay format that simultaneously encompasses as many of the relevant cellular processes as possible. In this report, the authors describe the configuration of a JMJD3 high-content cellular mechanistic imaging assay that uses single-cell multiparameter measurements to accurately assess cellular viability and the enzyme-dependent demethylation of the H3K27(Me)3 mark by exogenously expressed JMJD3. This approach couples robust statistical analyses with the spatial resolving power of cellular imaging. This enables segregation of expressing and nonexpressing cells into discrete subpopulations and consequently pharmacological quantification of compounds of interest in the expressing population at varying JMJD3 expression levels. Moreover, the authors demonstrate the utility of this hit identification strategy through the successful prosecution of a medium-throughput focused campaign of an 87 500-compound file, which has enabled the identification of JMJD3 cellular-active chemotypes. This study represents the first report of a demethylase high-content imaging assay with the ability to capture a repertoire of pharmacological tools, which are likely both to inform our mechanistic understanding of how JMJD3 is modulated and, more important, to contribute to the identification of novel therapeutic modalities for this demethylase enzyme.     

Computational Approaches and Tools Used in Identification of Dispersed Repetitive DNA Sequences

It has become clear that dispersed repeat sequences have played multiple roles in eukaryotic genome evolution including increasing genetic diversity through mutation, inducing changes in gene expression, and facilitating generation of novel genes. Growing recognition of the importance of dispersed repeats has fueled development of computational tools designed to expedite discovery and classification of repeats. Here we review major existing repeat exploration tools and discuss the algorithms utilized by these tools. Special attention is devoted to ab initio programs, i.e., those tools that do not rely upon previously identified repeats to find new repeat elements. We conclude by discussing the strengths and weaknesses of current tools and highlighting additional approaches that may advance repeat discovery/characterization.