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.

NRH:quinone oxidoreductase 2 (NQO2) is a flavoprotein that protects cells against radiation and chemical-induced oxidative stress. Disruption of the NQO2 gene in mice leads to gamma radiation-induced myeloproliferative diseases. In this report, we showed that the 20 S proteasome and NQO2 both interact with myeloid differentiation factor CCAAT-enhancer-binding protein alpha (C/EBPalpha). The interaction of the 20 S proteasome with C/EBPalpha led to the degradation of C/EBPalpha. NQO2, in the presence of its cofactor NRH, protected C/EBPalpha against 20 S degradation. Deletion and site-directed mutagenesis demonstrated that NQO2 and 20 S competed for the same binding region of S(268)GAGAGKAKKSV(279) in C/EBPalpha. Exposure of mice and HL-60 cells to gamma radiation enhanced the levels of NQO2, which led to an increased NQO2 interaction with C/EBPalpha and decreased 20 S interaction with C/EBPalpha. NQO2 stabilization of C/EBPalpha was independent of NQO1, even though both interacted with the same C/EBPalpha domain. NQO2(-/-) mice, deficient in NQO2, failed to stabilize C/EBPalpha. This contributed to the development of gamma radiation-induced myeloproliferative disease in NQO2(-/-) mice.

MicroRNAs (miRNAs) regulate gene expression by binding to their targets and promoting RNA degradation and/or inhibiting protein translation. In recent years, miRNAs have revolutionized our understanding of gene regulatory networks, providing new prospective tools to manage disease. Atherosclerosis and other cardiovascular diseases are a leading cause of disability and death in the US and in other western populations and pose an enormous burden on our healthcare system. Altered lipid homeostasis in liver or in the artery wall, and disruption of endothelial and smooth muscle cell function have been shown to contribute to the onset and progression of cardiovascular disease. This review focuses on recent advances in the field of vascular biology- and lipid metabolism-related miRNomics.

During an egg-laying cycle, oviparous animals transfer massive amounts of triglycerides, the major lipid component of very low density lipoprotein (VLDL), from the liver to the developing oocytes. A major stimulus for this process is the rise in estrogen associated with the onset of an egg-laying cycle. In mammals, the microsomal triglyceride transfer protein (MTP) is required for VLDL assembly and secretion. To enable studies to determine if MTP plays a role in basal and estrogen-stimulated VLDL assembly and secretion in an oviparous vertebrate, we have cloned and sequenced the chicken MTP cDNA. This cDNA encodes a protein of 893 amino acids with an N-terminal signal sequence. The primary sequence of chicken MTP is, on average, 65% identical to that of mammalian homologs, and 23% identical to the Drosophila melanogaster protein. We have obtained a clone of chicken embryo fibroblast cells that stably express the avian MTP cDNA and show that these cells display MTP activity as measured by the transfer of a fluorescently labeled neutral lipid. As in mammals, chicken MTP is localized to the endoplasmic reticulum as revealed by indirect immunofluorescence and by the fact that its N-linked oligosaccharide moiety remains sensitive to endoglycosidase H. Endogenous, enzymatically active MTP is also expressed in an estrogen receptor-expressing chicken hepatoma cell line that secretes apolipoprotein B-containing lipoproteins. In this cell line and in vivo, the expression and activity of MTP are not influenced by estrogen. Therefore, up-regulation of MTP in the liver is not required for the increased VLDL assembly during egg production in the chicken. This indicates that MTP is not rate-limiting, even for the massive estrogen-induced secretion of VLDL accompanying an egg-laying cycle.

Wingless acts as a morphogen in Drosophila wing discs, where it specifies cell fates and controls growth several cell diameters away from its site of expression. Thus, despite being acylated and membrane associated, Wingless spreads in the extracellular space. Recent studies have focussed on identifying the route that Wingless follows in the secretory pathway and determining how it is packaged for release. We have found that, in medium conditioned by Wingless-expressing Drosophila S2 cells, Wingless is present on exosome-like vesicles and that this fraction activates signal transduction. Proteomic analysis shows that Wingless-containing exosome-like structures contain many Drosophila proteins that are homologous to mammalian exosome proteins. In addition, Evi, a multipass transmembrane protein, is also present on exosome-like vesicles. Using these exosome markers and a cell-based RNAi assay, we found that the small GTPase Rab11 contributes significantly to exosome production. This finding allows us to conclude from in vivo Rab11 knockdown experiments, that exosomes are unlikely to contribute to Wingless secretion and gradient formation in wing discs. Consistent with this conclusion, extracellularly tagged Evi expressed from a Bacterial Artificial Chromosome is not released from imaginal disc Wingless-expressing cells.

Rab24 is an atypical member of the Rab GTPase family whose distribution in interphase cells has been characterized; however, its function remains largely unknown. In this study, we have analyzed the distribution of Rab24 throughout cell division. We have observed that Rab24 was located at the mitotic spindle in metaphase, at the midbody during telophase and in the furrow during cytokinesis. We have also observed partial co-localization of Rab24 and tubulin and demonstrated its association to microtubules. Interestingly, more than 90% of transiently transfected HeLa cells with Rab24 presented abnormal nuclear connections (i.e., chromatin bridges). Furthermore, in CHO cells stably transfected with GFP-Rab24wt, we observed a large percentage of binucleated and multinucleated cells. In addition, these cells presented an extremely large size and multiple failures in mitosis, as aberrant spindle formation (metaphase), delayed chromosomes (telophase) and multiple cytokinesis. A marked increase in binucleated, multinucleated and multilobulated nucleus formation was observed in HeLa cells depleted of Rab24. We also present evidence that a fraction of Rab24 associates with microtubules. In addition, Rab24 knock down resulted in misalignment of chromosomes and abnormal spindle formation in metaphase leading to the appearance of delayed chromosomes during late telophase and failures in cytokinesis. Our findings suggest that an adequate level of Rab24 is necessary for normal cell division. In summary, Rab24 modulates several mitotic events, including chromosome segregation and cytokinesis, perhaps through the interaction with microtubules.

Recent studies link synaptojanin 1 (synj1), the main phosphoinositol (4,5)-biphosphate phosphatase (PI(4,5)P2-degrading enzyme) in the brain and synapses, to Alzheimer disease. Here we report a novel mechanism by which synj1 reversely regulates cellular clearance of amyloid-beta (Abeta). Genetic down-regulation of synj1 reduces both extracellular and intracellular Abeta levels in N2a cells stably expressing the Swedish mutant of amyloid precursor protein (APP). Moreover, synj1 haploinsufficiency in an Alzheimer disease transgenic mouse model expressing the Swedish mutant APP and the presenilin-1 mutant DeltaE9 reduces amyloid plaque load, as well as Abeta40 and Abeta42 levels in hippocampus of 9-month-old animals. Reduced expression of synj1 attenuates cognitive deficits in these transgenic mice. However, reduction of synj1 does not affect levels of full-length APP and the C-terminal fragment, suggesting that Abeta generation by beta- and gamma-secretase cleavage is not affected. Instead, synj1 knockdown increases Abeta uptake and cellular degradation through accelerated delivery to lysosomes. These effects are partially dependent upon elevated PI(4,5)P2 with synj1 down-regulation. In summary, our data suggest a novel mechanism by which reduction of a PI(4,5)P2-degrading enzyme, synj1, improves amyloid-induced neuropathology and behavior deficits through accelerating cellular Abeta clearance.

Reconstructing early evolutionary events like the origins of informational and operational genes, membranes, and photophosphorylation is difficult because early evolutionary events can be masked by subsequent gene flows. Furthermore, as evolution progresses through both Darwinian survival of the fittest (tree-like evolution) and symbiotic/endosymbiotic cooperation (ring-like evolution), trees alone are not adequate to represent Earth's evolutionary history. Here, we reconstruct and root the New Rings of Life and use it as a framework for interpreting early events in the evolution of life. Unlike the three-domain hypothesis, the rings do not fit all life into one of three immutable categories, but rather accommodate new gene flows as novel organisms are discovered. A draft of the Rooted Rings of Life is reconstructed by analyzing the phylogenetic distributions of indels (insertions/deletions) and genes coding for fundamental molecular processes. Their phylogenetic distributions are inconsistent with all trees. Hypergeometric distribution analyses of them strongly localize the root of the rings to a segment of the deepest ring (P < 10(-21) and P < 10(-194)), and whole-genome analyses independently confirm the topology of the rooted rings (P < 7.1 x 10(-6)). The rings identify several large gene flows, including a flow of a thousand genes into the Halobacteria and the Eubacteria, the related photocyte flow, the flow of genes into the last common ancestor of the eocytes and the eukaryotes, and the informational and operational gene flows into the eukaryotes. The rooted rings also chronologically order steps in the evolution of extant taxa, that is, phototrophy evolved from Halobacteria (photophosphorylation) --> Heliobacteria (photosynthesis) --> Cyanobacteria (oxygenic photosynthesis).

Sonic hedgehog (SHH), a key regulator of embryonic neurogenesis, signals directly to neural stem cells (NSCs) in the subventricular zone (SVZ) and to astrocytes in the adult mouse forebrain. The specific mechanism by which the GLI2 and GLI3 transcriptional activators (GLI2(A) and GLI3(A)) and repressors (GLI2(R) and GLI3(R)) carry out SHH signaling has not been addressed. We found that the majority of slow-cycling NSCs express Gli2 and Gli3, whereas Gli1 is restricted ventrally and all three genes are downregulated when NSCs transition into proliferating progenitors. Surprisingly, whereas conditional ablation of Smo in postnatal glial fibrillary acidic protein-expressing cells results in cell-autonomous loss of NSCs and a progressive reduction in SVZ proliferation, without an increase in glial cell production, removal of Gli2 or Gli3 does not alter adult SVZ neurogenesis. Significantly, removing Gli3 in Smo conditional mutants largely rescues neurogenesis and, conversely, expression of a constitutive GLI3(R) in the absence of normal Gli2 and Gli3 abrogates neurogenesis. Thus unattenuated GLI3(R) is a primary inhibitor of adult SVZ NSC function. Ablation of Gli2 and Gli3 revealed a minor role for GLI2(R) and little requirement for GLI(A) function in stimulating SVZ neurogenesis. Moreover, we found that similar rules of GLI activity apply to SHH signaling in regulating SVZ-derived olfactory bulb interneurons and maintaining cortical astrocyte function. Namely, fewer superficial olfactory bulb interneurons are generated in the absence of Gli2 and Gli3, whereas astrocyte partial gliosis results from an increase in GLI3(R). Thus precise titration of GLI(R) levels by SHH is critical to multiple functions of adult NSCs and astrocytes.