Faculty Bibliography

In human cells, cytosolic citrate is a chief precursor for the synthesis of fatty acids, triacylglycerols, cholesterol and low-density lipoprotein. Cytosolic citrate further regulates the energy balance of the cell by activating the fatty-acid-synthesis pathway while downregulating both the glycolysis and fatty-acid beta-oxidation pathways. The rate of fatty-acid synthesis in liver and adipose cells, the two main tissue types for such synthesis, correlates directly with the concentration of citrate in the cytosol, with the cytosolic citrate concentration partially depending on direct import across the plasma membrane through the Na(+)-dependent citrate transporter (NaCT). Mutations of the homologous fly gene (Indy; I'm not dead yet) result in reduced fat storage through calorie restriction. More recently, Nact (also known as Slc13a5)-knockout mice have been found to have increased hepatic mitochondrial biogenesis, higher lipid oxidation and energy expenditure, and reduced lipogenesis, which taken together protect the mice from obesity and insulin resistance. To understand the transport mechanism of NaCT and INDY proteins, here we report the 3.2 A crystal structure of a bacterial INDY homologue. One citrate molecule and one sodium ion are bound per protein, and their binding sites are defined by conserved amino acid motifs, forming the structural basis for understanding the specificity of the transporter. Comparison of the structures of the two symmetrical halves of the transporter suggests conformational changes that propel substrate translocation.

Axons must switch responsiveness to guidance cues during development for correct pathfinding. Sonic Hedgehog (Shh) attracts spinal cord commissural axons ventrally toward the floorplate. We show that after crossing the floorplate, commissural axons switch their response to Shh from attraction to repulsion, so that they are repelled anteriorly by a posterior-high/anterior-low Shh gradient along the longitudinal axis. This switch is recapitulated in vitro with dissociated commissural neurons as they age, indicating that the switch is intrinsic and time dependent. 14-3-3 protein inhibition converted Shh-mediated repulsion of aged dissociated neurons to attraction and prevented the correct anterior turn of postcrossing commissural axons in vivo, an effect mediated through PKA. Conversely, overexpression of 14-3-3 proteins was sufficient to drive the switch from Shh-mediated attraction to repulsion both in vitro and in vivo. Therefore, we identify a 14-3-3 protein-dependent mechanism for a cell-intrinsic temporal switch in the polarity of axon turning responses.

Rationale: Although statins have been shown to have anti-inflammatory pleiotropic effects in experimental studies, the poor plaque targeting of orally administered statins limits their direct anti-inflammatory therapeutic effect. To that end, we have developed a simvastatin loaded high-density lipoprotein nanoparticle ([S]-rHDL), which has an improved plaque bioavailability and therefore exerts a higher therapeutic effect in apolipoprotein E knockout (ApoE KO) mice than orally administered simvastatin. The purpose of the current study is to understand the mechanism of this potent anti-inflammatory effect. Methods and Results: [S]-rHDL was found to specifically target plaque macrophages by fluorescence microscopy (a). To investigate the targeting efficiency of [S]-rHDL to monocytes/macrophages, we intravenously injected [S]-rHDL in ApoE KO mice (n=3/time point) and analyzed the cells from aortas and blood by flow cytometry. [S]-rHDL was found to target macrophages and monocytes in aortas (b), and to target inflammatory Gr-1hi monocytes more efficiently than anti-inflammatory Gr-1lo monocytes in blood (c). Last, laser capture microdissection was used to isolate plaque macrophages (n=7), and quantitative RT-PCR was used to measure their mRNA level of TNF-alpha, the hallmark of macrophage inflammation, which was significantly reduced (d). All the above data support our hypothesis that [S]-rHDL acts on inflammatory monocytes and plaque macrophages and thereby exerts a strong anti-inflammatory effect on atherosclerotic plaque. Conclusion: In ApoE KO mice, [S]-rHDL specifically targets plaque macrophages. [S]-rHDL also locally acts on macrophages in plaque and preferentially targets pro-inflammatory monocytes in blood, which results in a strong anti-inflammatory effect. This nanotherapy may represent a potent addition to the current standard of care for atherosclerosis patients

Painful bladder syndrome (PBS), or interstitial cystitis, is a poorly understood chronic disease that is characterized by thinning of the bladder epithelium and intense pain. Here we demonstrate that NAD(P)H:quinone oxidoreductase 1(-/-) (NQO1(-/-)) mice developed in our laboratory represent a new animal model of PBS. NQO1 is known to protect against physiological stress as well as protecting transcription factors against proteasomal degradation. In this study we demonstrate that NQO1 is necessary for bladder epithelium integrity and to prevent the development/progression of PBS. We observed downregulation of energy metabolism, adhesion, and apoptotic signaling cascades, which led to mitochondrial aberrations and profound alterations in energy metabolism, increased susceptibility to reactive oxygen species generation, and apoptosis in luminal epithelium in NQO1(-/-) mice that were absent in wild-type mice. These pathophysiological changes led to the incidence of PBS in NQO1(-/-) mice. Altogether, the results demonstrate for the first time that NQO1 is an endogenous factor in protection against PBS.

To identify molecular mechanisms that function in G-protein signaling, we have performed molecular genetic studies of a simple behavior of the nematode Caenorhabditis elegans, egg laying, which is driven by a pair of serotonergic neurons, the hermaphrodite-specific neurons (HSNs). The activity of the HSNs is regulated by the G(o)-coupled receptor EGL-6, which mediates inhibition of the HSNs by neuropeptides. We report here that this inhibition requires one of three inwardly rectifying K(+) channels encoded by the C. elegans genome: IRK-1. Using ChannelRhodopsin-2-mediated stimulation of HSNs, we observed roles for egl-6 and irk-1 in regulating the excitability of HSNs. Although irk-1 is required for inhibition of HSNs by EGL-6 signaling, we found that other G(o) signaling pathways that inhibit HSNs involve irk-1 little or not at all. These findings suggest that the neuropeptide receptor EGL-6 regulates the potassium channel IRK-1 via a dedicated pool of G(o) not involved in other G(o)-mediated signaling. We conclude that G-protein-coupled receptors that signal through the same G-protein in the same cell might activate distinct effectors and that specific coupling of a G-protein-coupled receptor to its effectors can be determined by factors other than its associated G-proteins.

Staphylococcus aureus peptidoglycan (PG) is densely functionalized with anionic polymers called wall teichoic acids (WTAs). These polymers contain three tailoring modifications: d-alanylation, alpha-O-GlcNAcylation, and beta-O-GlcNAcylation. Here we describe the discovery and biochemical characterization of a unique glycosyltransferase, TarS, that attaches beta-O-GlcNAc (beta-O-N-acetyl-D-glucosamine) residues to S. aureus WTAs. We report that methicillin resistant S. aureus (MRSA) is sensitized to beta-lactams upon tarS deletion. Unlike strains completely lacking WTAs, which are also sensitive to beta-lactams, DeltatarS strains have no growth or cell division defects. Because neither alpha-O-GlcNAc nor beta-O-Glucose modifications can confer resistance, the resistance phenotype requires a highly specific chemical modification of the WTA backbone, beta-O-GlcNAc residues. These data suggest beta-O-GlcNAcylated WTAs scaffold factors required for MRSA resistance. The beta-O-GlcNAc transferase identified here, TarS, is a unique target for antimicrobials that sensitize MRSA to beta-lactams.

Blood vessels deliver oxygen, nutrients, hormones and immunity factors throughout the body. To perform these vital functions, vascular cords branch, lumenize and interconnect. Yet, little is known about the cellular, molecular and physiological mechanisms that control how circulatory networks form and interconnect. Specifically, how circulatory networks merge by interconnecting in parallel along their boundaries remains unexplored. To examine this process we studied the formation and functional maturation of the plexus that forms between the Dorsal Longitudinal Anastomotic Vessels (DLAVs) in the zebrafish. We find that the migration and proliferation of endothelial cells within the DLAVs and their Segmental (Se) vessel precursors drives DLAV plexus formation. Remarkably, the presence of Se vessels containing only endothelial cells of the arterial lineage is sufficient for DLAV plexus morphogenesis, suggesting that endothelial cells from the venous lineage make a dispensable or null contribution to this process. The discovery of a circuit that integrates the inputs of circulatory flow and Vascular Endothelial Growth Factor (VEGF) signaling to modulate aortic arch angiogenesis, together with the expression of components of this circuit in the trunk vasculature, prompted us to interrogate the role of these inputs and their relationship during DLAV plexus formation. We find that circulatory flow and VEGF signaling make additive contributions to DLAV plexus morphogenesis, rather than acting as essential inputs with equivalent contributions as they do during aortic arch angiogenesis. Our observations underscore the existence of context-dependent differences in the integration of physiological stimuli and signaling cascades during vascular development.

ML-IAP [melanoma IAP (inhibitor of apoptosis)] is an anti-apoptotic protein that is expressed highly in melanomas where it contributes to resistance to apoptotic stimuli. The anti-apoptotic activity and elevated expression of IAP family proteins in many human cancers makes IAP proteins attractive targets for inhibition by cancer therapeutics. Small-molecule IAP antagonists that bind with high affinities to select BIR (baculovirus IAP repeat) domains have been shown to stimulate auto-ubiquitination and rapid proteasomal degradation of c-IAP1 (cellular IAP1) and c-IAP2 (cellular IAP2). In the present paper, we report ML-IAP proteasomal degradation in response to bivalent, but not monovalent, IAP antagonists. This degradation required ML-IAP ubiquitin ligase activity and was independent of c-IAP1 or c-IAP2. Although ML-IAP is best characterized in melanoma cells, we show that ML-IAP expression in normal mammalian tissues is restricted largely to the eye, being most abundant in ciliary body epithelium and retinal pigment epithelium. Surprisingly, given this pattern of expression, gene-targeted mice lacking ML-IAP exhibited normal intraocular pressure as well as normal retinal structure and function. The results of the present study indicate that ML-IAP is dispensable for both normal mouse development and ocular homoeostasis.

Radiation therapy has the potential to convert the tumor into an in situ individualized vaccine by inducing immunogenic cancer cell death and pro-inflammatory cytokines and chemokines; however this potential is rarely realized by irradiation alone. We hypothesized that radiation-induced immunosuppressive factors may hinder its pro-immunogenic effects. Transforming growth factor beta (TGFbeta) has immunosuppressive function for dendritic cells and T cells and is activated by radiation. Here we tested the hypothesis that inhibiting TGFbeta during radiation treatment would induce an immunogenic response. Poorly immunogenic, highly metastatic 4T1 carcinoma cells were injected s.c. in syngeneic BALB/c mice (day 0). TGFbeta neutralizing 1D11 or isotype control 13C4 monoclonal antibodies were given i.p. (200 mg/mouse) every other day from day 12 to 28. Tumors were irradiated with 6Gy on five consecutive days beginning on day 13. Tumor growth was measured consecutively. Mice were euthanized at day 21 for analysis, at day 28 for enumeration of lung metastases, or followed for survival. Gene expression profiles were obtained using Affymetrix mouse genome 430 2.0 array. Tumor growth rates and the frequency of lung metastases were similar in mice receiving control antibody or 1D11 alone. Radiation treatment caused significant (P=0.0065) tumor growth delay but did not inhibit lung metastases. In contrast, mice treated with both 1D11 and radiation exhibited significantly greater tumor growth control and reduced lung metastases (P<0.0001), and significantly prolonged survival (P<0.005). As expected, TGFbeta signalling was inhibited with 1D11 as measured in CD4+ and CD8+ T cells from tumor-draining lymph nodes at day 21. CD8+ T cells producing IFN in response to a tumor-specific antigen were detected only in mice treated with 1D11 and radiation. Expression profiles showed that genes associated with immune response and T cell activation were upregulated in irradiated tumors of mice treated with 1D11 compared to other treatment groups. In vivo depletion experiments demonstrated that T cells were essential for the improved tumor control and inhibition of lung metastases of mice treated with 1D11 and radiation. These data support a critical role for TGFbeta as a regulator of the pro-immunogenic effects of local tumor radiotherapy. Inhibition of TGFbeta during radiotherapy may promote self-immunization and achieve systemic control of metastatic disease. Supported by DOD BCRP grant BC100481P2