Faculty Bibliography

Drosophila olfactory sensory neurons express either odorant receptors or ionotropic glutamate receptors (IRs). The sensory neurons that express IR64a, a member of the IR family, send axonal projections to either the DC4 or DP1m glomeruli in the antennal lobe. DC4 neurons respond specifically to acids/protons, whereas DP1m neurons respond to a broad spectrum of odorants. The molecular composition of IR64a-containing receptor complexes in either DC4 or DP1m neurons is not known, however. Here, we immunoprecipitated the IR64a protein from lysates of fly antennal tissue and identified IR8a as a receptor subunit physically associated with IR64a by mass spectrometry. IR8a mutants and flies in which IR8a was knocked down by RNAi in IR64a+ neurons exhibited defects in acid-evoked physiological and behavioral responses. Furthermore, we found that the loss of IR8a caused a significant reduction in IR64a protein levels. When expressed in Xenopus oocytes, IR64a and IR8a formed a functional ion channel that allowed ligand-evoked cation currents. These findings provide direct evidence that IR8a is a subunit that forms a functional olfactory receptor with IR64a in vivo to mediate odor detection.

Although the role of E proteins in the thymocyte development is well documented, much less is known about their function in peripheral T cells. Here we demonstrated that CD4 promoter-driven transgenic expression of Id1, a naturally occurring dominant-negative inhibitor of E proteins, can substitute for the co-stimulatory signal delivered by CD28 to facilitate the proliferation and survival of naive CD4+ cells upon anti-CD3 stimulation. We next discovered that IL-2 production and NF-kappaB activity after anti-CD3 stimulation were significantly elevated in Id1-expressing cells, which may be, at least in part, responsible for the augmentation of their proliferation and survival. Taken together, results from this study suggest an important role of E and Id proteins in peripheral T cell activation. The ability of Id proteins to by-pass co-stimulatory signals to enable T cell activation has significant implications in regulating T cell immunity.

Lymphocyte homing, which contributes to inflammation, has been studied extensively in the small intestine, but there is little known about homing to the large intestine, the site most commonly affected in inflammatory bowel disease. GPR15, an orphan G protein-coupled receptor, controlled the specific homing of T cells, particularly FOXP3+ regulatory T cells (Tregs), to the large intestine lamina propria (LILP). GPR15 expression was modulated by gut microbiota and transforming growth factor-beta1, but not by retinoic acid. GPR15-deficient mice were prone to develop more severe large intestine inflammation, which was rescued by the transfer of GPR15-sufficient Tregs. Our findings thus describe a T cell homing receptor for LILP and indicate that GPR15 plays a role in mucosal immune tolerance largely by regulating the influx of Tregs.

PGRN and its derived engineered protein, Atsttrin, were reported to antagonize TNFalpha and protect against inflammatory arthritis [Tang, W. et al. (2011) The growth factor progranulin binds to TNF receptors and is therapeutic against inflammatory arthritis in mice. Science 332 (6028) 478-484]. Here we found that PGRN level was also significantly elevated in skin inflammation. PGRN-/- mice exhibited more severe inflammation following induction of oxazolone (OXA). In contrast, recombinant Atsttrin protein effectively attenuated inflammation in mice dermatitis model. In addition, the protective role of PGRN and Atsttrin in dermatitis was probably due to their inhibition on NF-kappaB signaling. Collectively, PGRN, especially its derived engineered protein, Atsttrin, may represent a potential molecular target for prevention and treatment of inflammatory skin diseases.

We have previously reported enhancing the imaging of atherosclerotic plaques in mice using reconstituted high density lipoproteins (HDL) as nanocarriers for the MRI contrast agent gadolinium (Gd). This study focuses on the underlying mechanisms of Gd delivery to atherosclerotic plaques. HDL, LDL, and VLDL particles containing Gd chelated to phosphatidyl ethanolamine (DTPA-DMPE) and a lipidic fluorophore were used to demonstrate the transfer of Gd-phospholipids among plasma lipoproteins in vitro and in vivo. To determine the basis of this transfer, the roles of phospholipid transfer protein (PLTP) and lipoprotein lipase (LpL) in mediating the migration of Gd-DTPA-DMPE among lipoproteins were investigated. The results indicated that neither was an important factor, suggesting that spontaneous transfer of Gd-DTPA-DMPE was the most probable mechanism. Finally, two independent mouse models were used to quantify the relative contributions of HDL and LDL reconstituted with Gd-DTPA-DMPE to plaque imaging enhancement by MR. Both sets of results suggested that Gd-DTPA-DMPE originally associated with LDL was about twice as effective as that injected in the form of Gd-HDL, and that some of Gd-HDL's effectiveness in vivo is indirect through transfer of the imaging agent to LDL. In conclusion, the fate of Gd-DTPA-DMPE associated with a particular type of lipoprotein is complex, and includes its transfer to other lipoprotein species that are then cleared from the plasma into tissues.

Selected Reaction Monitoring (SRM) is a method of choice for accurate quantitation of low-abundance proteins in complex backgrounds. This strategy is, however, sensitive to interference from other components in the sample that have the same precursor and fragment masses as the monitored transitions. We present here an approach to detect interference by using the expected relative intensity of SRM transitions. We also designed an algorithm to automatically detect the linear range of calibration curves. These approaches were applied to the experimental data of Clinical Proteomic Tumor Analysis Consortium (CPTAC) Verification Work Group Study 7 and show that the corrected measurements provide more accurate quantitation than the uncorrected data.

Voltage-gated K(+) (Kv) channels are molecular switches that sense membrane potential and in response open to allow K(+) ions to diffuse out of the cell. In these proteins, sensor and pore belong to two distinct structural modules. We previously showed that the pore module alone is a robust yet dynamic structural unit in lipid membranes and that it senses potential and gates open to conduct K(+) with unchanged fidelity. The implication is that the voltage sensitivity of K(+) channels is not solely encoded in the sensor. Given that the coupling between sensor and pore remains elusive, we asked whether it is then possible to convert a pore module characterized by brief openings into a conductor with a prolonged lifetime in the open state. The strategy involves selected probes targeted to the filter gate of the channel aiming to modulate the probability of the channel being open assayed by single channel recordings from the sensorless pore module reconstituted in lipid bilayers. Here we show that the premature closing of the pore is bypassed by association of the filter gate with two novel open conformation stabilizers: an antidepressant and a peptide toxin known to act selectively on Kv channels. Such stabilization of the conductive conformation of the channel is faithfully mimicked by the covalent attachment of fluorescein at a cysteine residue selectively introduced near the filter gate. This modulation prolongs the occupancy of permeant ions at the gate. It is this longer embrace between ion and gate that we conjecture underlies the observed stabilization of the conductive conformation. This study provides a new way of thinking about gating.

Rationale: Foam cell formation due to excessive accumulation of cholesterol by macrophages is a pathological hallmark of atherosclerosis, the major cause of morbidity and mortality in Western societies. Liver X nuclear receptors (LXRs) regulate the expression of the adenosine triphosphate-binding cassette (ABC) transporters, including ABCA1 and ABCG1. ABCA1 and ABCG1 facilitate the efflux of cholesterol from macrophages and regulate high-density lipoprotein (HDL) biogenesis. Increasing evidence supports the role of microRNA (miRNAs) in regulating cholesterol metabolism through ABC transporters. Objective: We aimed to identify novel miRNAs that regulate cholesterol metabolism in macrophages stimulated with LXR agonists. Methods and Results: To map the miRNA expression signature of macrophages stimulated with LXR agonists, we performed a miRNA profiling microarray analysis in primary mouse peritoneal macrophages stimulated with LXR ligands. We report that LXR ligands increase miR-144 expression in macrophages and mouse livers. Overexpression of miR-144 reduces ABCA1 expression and attenuates cholesterol efflux to ApoA1 in macrophages. Delivery of miR-144 oligonucleotides to mice attenuates ABCA1 expression in the liver, reducing HDL levels. Conversely, silencing of miR-144 in mice increases the expression of ABCA1 and plasma HDL levels. Thus, miR-144 appears to regulate both macrophage cholesterol efflux and HDL biogenesis in the liver. Conclusions: 1) miR-144 regulates cholesterol metabolism via suppressing ABCA1 expression; and 2) modulation of miRNAs may represent a potential therapeutical intervention for treating dyslipidemia and atherosclerotic vascular disease.

Cholesterol is a structural component of the cell and is indispensable for normal cellular function, although its excess often leads to abnormal proliferation, migration, inflammatory responses and/or cell death. To prevent cholesterol overload, ATP-binding cassette (ABC) transporters mediate cholesterol efflux from the cells to apolipoprotein A-I (apoA-I) and the apoA-I-containing high-density lipoprotein (HDL). Maintaining efficient cholesterol efflux is essential for normal cellular function. However, the role of cholesterol efflux in angiogenesis and the identity of its local regulators are poorly understood. Here we show that apoA-I binding protein (AIBP) accelerates cholesterol efflux from endothelial cells to HDL and thereby regulates angiogenesis. AIBP- and HDL-mediated cholesterol depletion reduces lipid rafts, interferes with VEGFR2 (also known as KDR) dimerization and signalling and inhibits vascular endothelial growth factor-induced angiogenesis in vitro and mouse aortic neovascularization ex vivo. Notably, Aibp, a zebrafish homologue of human AIBP, regulates the membrane lipid order in embryonic zebrafish vasculature and functions as a non-cell-autonomous regulator of angiogenesis. aibp knockdown results in dysregulated sprouting/branching angiogenesis, whereas forced Aibp expression inhibits angiogenesis. Dysregulated angiogenesis is phenocopied in Abca1 (also known as Abca1a) Abcg1-deficient embryos, and cholesterol levels are increased in Aibp-deficient and Abca1 Abcg1-deficient embryos. Our findings demonstrate that secreted AIBP positively regulates cholesterol efflux from endothelial cells and that effective cholesterol efflux is critical for proper angiogenesis.

The chromatin state of pluripotency genes has been studied extensively in embryonic stem cells (ESCs) and differentiated cells, but their potential interactions with other parts of the genome remain largely unexplored. Here, we identified a genome-wide, pluripotency-specific interaction network around the Nanog promoter by adapting circular chromosome conformation capture sequencing. This network was rearranged during differentiation and restored in induced pluripotent stem cells. A large fraction of Nanog-interacting loci were bound by Mediator or cohesin in pluripotent cells. Depletion of these proteins from ESCs resulted in a disruption of contacts and the acquisition of a differentiation-specific interaction pattern prior to obvious transcriptional and phenotypic changes. Similarly, the establishment of Nanog interactions during reprogramming often preceded transcriptional upregulation of associated genes, suggesting a causative link. Our results document a complex, pluripotency-specific chromatin "interactome" for Nanog and suggest a functional role for long-range genomic interactions in the maintenance and induction of pluripotency.