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Acylation of proteins with fatty acids is important for the regulation of membrane association, trafficking, subcellular localization, and activity of many cellular proteins. While significant progress has been made in our understanding of the two major forms of protein acylation with fatty acids, N-myristoylation and S-palmitoylation, studies of the acylation of lysine residues, within proteins, with fatty acids have lagged behind. Demonstrated here is the use of integrative chemical biology approaches to examine human sirtuins as de-fatty-acid acylases invitro and in cells. Photo-crosslinking chemistry is used to investigate enzymes which recognize fatty-acid acylated lysine. Human Sirt2 was identified as a robust lysine de-fatty-acid acylase invitro. The results also show that Sirt2 can regulate the acylation of lysine residues, of proteins, with fatty acids within cells.

Host innate recognition triggers key immune responses for viral elimination. The sensing mechanism of hepatitis B virus (HBV), a DNA virus, and the subsequent downstream signaling events remain to be fully clarified. Here we found that type III but not type I interferons are predominantly induced in human primary hepatocytes in response to HBV infection, through retinoic acid-inducible gene-I (RIG-I)-mediated sensing of the 5'-epsilon region of HBV pregenomic RNA. In addition, RIG-I could also counteract the interaction of HBV polymerase (P protein) with the 5'-epsilon region in an RNA-binding dependent manner, which consistently suppressed viral replication. Liposome-mediated delivery and vector-based expression of this e region-derived RNA in liver abolished the HBV replication in human hepatocyte-chimeric mice. These findings identify an innate-recognition mechanism by which RIG-I dually functions as an HBV sensor activating innate signaling and to counteract viral polymerase in human hepatocytes.

The inability to visualize the true extent of cancers represents a significant challenge in many areas of oncology. The margins of most cancer types are not well demarcated because the cancer diffusely infiltrates the surrounding tissues. Furthermore, cancers may be multifocal and characterized by the presence of microscopic satellite lesions. Such microscopic foci represent a major reason for persistence of cancer, local recurrences, and metastatic spread, and are usually impossible to visualize with currently available imaging technologies. An imaging method to reveal the true extent of tumors is desired clinically and surgically. We show the precise visualization of tumor margins, microscopic tumor invasion, and multifocal locoregional tumor spread using a new generation of surface-enhanced resonance Raman scattering (SERRS) nanoparticles, which are termed SERRS nanostars. The SERRS nanostars feature a star-shaped gold core, a Raman reporter resonant in the near-infrared spectrum, and a primer-free silication method. In genetically engineered mouse models of pancreatic cancer, breast cancer, prostate cancer, and sarcoma, and in one human sarcoma xenograft model, SERRS nanostars enabled accurate detection of macroscopic malignant lesions, as well as microscopic disease, without the need for a targeting moiety. Moreover, the sensitivity (1.5 fM limit of detection) of SERRS nanostars allowed imaging of premalignant lesions of pancreatic and prostatic neoplasias. High sensitivity and broad applicability, in conjunction with their inert gold-silica composition, render SERRS nanostars a promising imaging agent for more precise cancer imaging and resection.

Sterols are a critical component of cell membranes of eukaryotes. In mammalian cells there is approximately a six-fold range in the cholesterol content in various organelles. The cholesterol content of membranes plays an important role in organizing membranes for signal transduction and protein trafficking as well as in modulating the physiochemical properties of membranes. Cholesterol trafficking among organelles is highly dynamic and is mediated by both vesicular and non-vesicular processes. Several proteins have been proposed to mediate inter-organelle trafficking of cholesterol. However, several aspects of the mechanisms involved in regulating trafficking and distribution of cholesterol remain to be elucidated. In the present chapter, we discuss the cellular mechanisms involved in cholesterol distribution and the trafficking processes involved in maintaining sterol homoeostasis.

Arc is a unique immediate early gene whose expression is induced as synapses are being modified during learning. The uniqueness comes from the fact that newly synthesized Arc mRNA is rapidly transported throughout dendrites where it localizes near synapses that were recently activated. Here, we summarize aspects of Arc mRNA translation in dendrites in vivo, focusing especially on features of its expression that are paradoxical or that donot fit in with current models of how Arc protein operates. Findings from in vivo studies that donot quite fit include: (1) Following induction of LIP in vivo, Arc mRNA and protein localize near active synapses, but are also distributed throughout dendrites. In contrast, Arc mRNA localizes selectively near active synapses when stimulation is continued as Arc mRNA is transported into dendrites; (2) Strong induction of Arc expression as a result of a seizure does not lead to a rundown of synaptic efficacy in vivo as would be predicted by the hypothesis that high levels of Arc cause glutamate receptor endocytosis and LTD. (3) Arc protein is synthesized in the perinuclear cytoplasm rapidly after transcriptional activation, indicating that at least a pool of Arc mRNA is not translationally repressed to allow for dendritic delivery; (4) Increases in Arc mRNA in dendrites are not paralleled by increases in levels of exon junction complex (EJC) proteins. These results of studies of mRNA trafficking in neurons in vivo provide a new perspective on the possible roles of Arc in activity-dependent synaptic modifications.

Delta-aminolevulinic acid dehydratase single nucleotide polymorphism 2 (ALAD(2)) and peptide transporter haplotype 2*2 (hPEPT2*2) through different pathways can increase brain levels of delta-aminolevulinic acid and are associated with higher blood lead burden in young children. Past child and adult findings regarding ALAD(2) and neurobehavior have been inconsistent, and the possible association of hPEPT2*2 and neurobehavior has not yet been examined. Mean blood lead level (BLL), genotype, and neurobehavioral function (fine motor dexterity, working memory, visual attention and short-term memory) were assessed in 206 males and 215 females ages 5.1-11.8 years. Ninety-six percent of children had BLLs < 5.0 mu g/dl. After adjusting for covariates (sex, age and mother's level of education) and sibling exclusion (N = 252), generalized linear mixed model analyses showed opposite effects for the ALAD(2) and hPEPT2*2 genetic variants. Significant effects for ALAD(2) were observed only as interactions with BLL and the results suggested that ALAD(2) was neuroprotective. As BLL increased, ALAD(2) was associated with enhanced visual attention and enhanced working memory (fewer commission errors). Independent of BLL, hPEPT2*2 predicted poorer motor dexterity and poorer working memory (more commission errors). BLL alone predicted poorer working memory from increased omission errors. The findings provided further substantiation that (independent of the genetic variants examined) lowest-level lead exposure disrupted early neurobehavioral function, and suggested that common genetic variants alter the neurotoxic potential of low-level lead. ALAD(2) and hPEPT2*2 may be valuable markers of risk, and indicate novel mechanisms of lead-induced neurotoxicity. Longitudinal studies are needed to examine long-term influences of these genetic variants on neurobehavior. (C) 2014 Elsevier Inc. All rights reserved.

Sterols are essential biological molecules in the majority of life forms. Sterol reductases(1) including Delta(14)-sterol reductase (C14SR, also known as TM7SF2), 7-dehydrocholesterol reductase (DHCR7) and 24-dehydrocholesterol reductase (DHCR24) reduce specific carbon-carbon double bonds of the sterol moiety using a reducing cofactor during sterol biosynthesis. Lamin B receptor(2) (LBR), an integral inner nuclear membrane protein, also contains a functional C14SR domain. Here we report the crystal structure of a Delta(14)-sterol reductase (MaSR1) from the methanotrophic bacterium Methylomicro-bium alcaliphilum 20Z (a homologue of human C14SR, LBR and DHCR7) with the cofactor NADPH. The enzyme contains ten transmembrane segments (TM1-10). Its catalytic domain comprises the carboxy-terminal half (containing TM6-10) and envelops two interconnected pockets, one of which faces the cytoplasm and houses NADPH, while the other one is accessible from the lipid bilayer. Comparison with a soluble steroid 5 beta-reductase structure(3) suggests that the reducing end of NADPH meets the sterol substrate at the juncture of the two pockets. A sterol reductase activity assay proves that MaSR1 can reduce the double bond of a cholesterol biosynthetic intermediate, demonstrating functional conservation to human C14SR. Therefore, our structure as a prototype of integral membrane sterol reductases provides molecular insight into mutations in DHCR7 and LBR for inborn human diseases.

Atopic dermatitis (AD) is characterized by type 2 helper T (Th2) cell-driven inflammation. Dupilumab is a fully human monoclonal antibody directed against the IL-4 receptor alpha subunit that blocks the signaling of IL-4 and IL-13, both key cytokines in Th2-mediated pathways. In Phase I and II studies of adults with moderate-to-severe AD, dupilumab administered as monotherapy or with topical corticosteroids resulted in rapid, significant improvements in clinical efficacy, patient-reported outcomes, and Th2-related serum and tissue biomarkers, and shifted the RNA expression profile of lesional skin to a more nonlesional signature. In all clinical studies to date, dupilumab has shown a favorable safety profile with no dose-limiting toxicity. The robust effects of dupilumab on skin inflammation and pruritus confirm the pathogenic role of IL-4 and IL-13 signaling in adult AD, and further support the application of Th2 cytokine antagonists in the treatment of this disease.

Elevated levels of thyroid hormones (TH) reduce estradiol (E2)-dependent female sexual behavior. E2 stimulates progesterone receptor (Pgr) and oxytocin receptor (Oxtr) within the ventromedial hypothalamus and preoptic area, critical hypothalamic nuclei for sexual and maternal behavior, respectively. Here, we investigated the impact of TH on E2-dependent transcriptional mechanisms in female mice. First, we observed that triiodothyronine (T-3) inhibited the E2 induction of Pgr and Oxtr. We hypothesized that differences in histone modifications and receptor recruitment could explain the influence of TH on E2-responsive Pgr and Oxtr expression. We observed that histone H3 acetylation (H3Ac) and methylation (H3K4me3) was gene and brain-region specific. We then analyzed the recruitment of estrogen receptor alpha (ER alpha) and TH receptor alpha (TR alpha) on the putative regulatory sequences of Pgr and Oxtr. Interestingly, T-3 inhibited E2-induced ER alpha binding to a specific Pgr enhancer site, whereas TR alpha binding was not affected, corroborating our theory that the competitive binding of TR alpha to an ER alpha binding site can inhibit ER alpha transactivation and the subsequent E2-responsive gene expression. On the Oxtr promoter, E2 and T-3 worked together to modulate ER alpha and TR alpha binding. Finally, the E2-dependent induction of cofactors was reduced by hypothyroidism and T-3. Thus, we determined that the Pgr and Oxtr promoter regions are responsive to E2 and that T-3 interferes with the E2 regulation of Pgr and Oxtr expression by altering the recruitment of receptors to DNA and changing the availability of cofactors. Collectively, our findings provide insights into molecular mechanisms of response to E2 and TH interactions controlling sex behavior in the hypothalamus. (C) 2015 S. Karger AG, Basel