ORP2 is a sterol receptor that regulates cellular lipid metabolism

ORP2 is a member of mammalian oxysterol binding protein (OSBP)-related protein/gene family (ORPs), which is found in almost every eukaryotic organism. ORPs have been suggested to participate in the regulation of cellular lipid metabolism, vesicle trafficking and cellular signaling. ORP2 is a cytosolic protein that is ubiquitously expressed and most abundant in the brain. In previous studies employing stable cell lines with constitutive ORP2 overexpression ORP2 was shown to affect cellular cholesterol metabolism. The aim of this study was to characterize the properties and function of ORP2 further. ORP2 ligands were searched for among sterols and phosphoinositides using purified ORP2 and in vitro binding assays. As expected, ORP2 bound several oxysterols and cholesterol, the highest affinity ligand being 22(R)hydroxycholesterol. In addition, affinity for anionic membrane phospholipids, phosphoinositides was observed, which may assist in the membrane targeting of ORP2. Intracellular localization of ORP2 was also investigated. ORP2 was observed on the surface of cytoplasmic lipid droplets, which are storage organelles for neutral lipids. Lipid droplet targeting of ORP2 was inhibited when 22(R)hydroxycholesterol was added to the cells or when the N-terminal FFAT-motif of ORP2 was mutated, suggesting that oxysterols and the N-terminus of ORP2 regulate the localization and the function of ORP2. The role of ORP2 in cellular lipid metabolism was studied using HeLa cell lines that can be induced to overexpress ORP2. Overexpression of ORP2 was shown to enhance cholesterol efflux from the cells resulting in a decreased amount of cellular free cholesterol. ORP2 overexpressing cells responded to the loss of cholesterol by upregulating cholesterol synthesis and uptake. Intriguingly, also cholesterol esterification was increased in ORP2 overexpressing cells. These results may be explained by the ability of ORP2 to bind and thus transport cholesterol, which most likely leads to changes in cholesterol metabolism when ORP2 is overexpressed. ORP2 function was further investigated by silencing the endogenous ORP2 expression with short interfering RNAs (siRNA) in A431 cells. Silencing of ORP2 led to a delayed break-down of triglycerides under lipolytic conditions and an increased amount of cholesteryl esters in the presence of excess triglycerides. Together these results suggest that ORP2 is a sterol-regulated protein that functions on the surface of cytoplasmic lipid droplets to regulate the metabolism of triglycerides and cholesteryl esters. Although the exact mode of ORP2 action still remains unclear, this study serves as a good basis to investigate the molecular mechanisms and possible cell type specific functions of ORP2.

Generation and Characterization of the Cation-Chloride Cotransporter KCC2 Hypomorphic Mouse

The cation-Cl- cotransporter (CCC) family comprises of Na+-Cl- cotransporter (NCC), Na+-K+-2Cl- cotransporters (NKCC1-2), and four K+-Cl- cotransporters (KCC1-4). These proteins are involved in several physiological activities, such as cell volume regulation. In neuronal tissues, NKCC1 and KCC2 are important in determining the intracellular Cl- levels and hence the neuronal responses to inhibitory neurotransmitters GABA and glycine. One aim of the work was to elucidate the roles for CCC isoforms in the control of nervous system development. KCC2 mRNA was shown to be developmentally up-regulated and follow neuronal maturation, whereas NKCC1 and KCC4 transcripts were highly expressed in the proliferative zones of subcortical regions. KCC1 and KCC3 mRNA displayed low expression throughout the embryogenesis. These expression profiles suggest a role for CCC isoforms in maturation of synaptic responses and in the regulation of neuronal proliferation during embryogenesis. The major aim of this work was to study the biological consequences of KCC2-deficiency in the adult CNS, by generating transgenic mice retaining 15-20% of normal KCC2 levels. In addition, by using these mice as a tool for in vivo pharmacological analysis, we investigated the requirements for KCC2 in tonic versus phasic GABAA receptor-mediated inhibition. KCC2-deficient mice displayed normal reproduction and life span, but showed several behavioral abnormalities, including increased anxiety-like behavior, impaired performance in water maze, alterations in nociceptive processing, and increased seizure susceptibility. In contrast, the mice displayed apparently normal spontaneous locomotor activity and motor coordination. Pharmacological analysis of KCC2-deficient mice revealed reduced sensititivity to diazepam, but normal gaboxadol-induced sedation, neurosteroid hypnosis and alcohol-induced motor impairment. Electrophysiological recordings from CA1-CA3 subregions of the hippocampus showed that KCC2 deficiency affected the reversal potentials of both the phasic and tonic GABA currents, and that the tonic conductance was not affected. The results suggest that requirement for KCC2 in GABAergic neurotransmission may differ among several functional systems in the CNS, which is possibly due to the more critical role of KCC2 activity in phasic compared to tonic GABAergic inhibition.

CD1 expression and CD1-restricted T cell activity in normal and tumour-bearing human liver

CD1d-restricted natural killer T (NKT) cells expressing invariant Valpha14Jalpha18 T cell receptor alpha-chains are abundant in murine liver and are implicated in the control of malignancy, infection and autoimmunity. Invariant NKT cells have potent anti-metastatic effects in mice and phase I clinical trials involving their homologues in humans are ongoing. However, invariant NKT cells are less abundant in human liver ( approximately 0.5% of hepatic T cells) than in murine liver (up to 50%) and it is not known if other hepatic T cells are CD1-restricted. We have examined expression of CD1a, CD1b, CD1c and CD1d mRNA and protein in human liver and evaluated the reactivity of mononuclear cells (MNC) from histologically normal and tumour-bearing human liver specimens against these CD1 isoforms. Messenger RNA for all CD1 isotypes was detectable in all liver samples. CD1c and CD1d were expressed at the protein level by hepatic MNC. CD1d, only, was detectable at the cell surface, but CD1c and CD1d were found at an intracellular location in significant numbers of liver MNC. CD1b was not expressed by MNC from healthy livers but was detectable within MNC in all tumour samples tested. Hepatic T cells exhibited reactivity against C1R cells expressing transfected CD1c and CD1d, but neither CD1a nor CD1b. These cells secreted interferon-gamma (IFN-gamma) but not interleukin-4 (IL-4) upon stimulation. In contrast, similar numbers of peripheral T cells released 13- and 16-fold less IFN-gamma in response to CD1c and CD1d, respectively. CD1c and CD1d expression and T cell reactivity were not altered in tumour-bearing liver specimens compared to histologically normal livers. These data suggest that, in addition to invariant CD1d-restricted NKT cells, autoreactive T cells that recognise CD1c and CD1d and release inflammatory cytokines are abundant in human liver.

Regulation of extracellular matrix genes by arecoline in primary gingival fibroblasts requires epithelial factors

Background and Objective: Oral submucous fibrosis, a disease of collagen disorder, has been attributed to arecoline present in the saliva of betel quid chewers. However, the molecular basis of the action of arecoline in the pathogenesis of oral submucous fibrosis is poorly understood. The basic aim of our study was to elucidate the mechanism underlying the action of arecoline on the expression of genes in oral fibroblasts. Material and Methods: Human keratinocytes (HaCaT cells) and primary human gingival fibroblasts were treated with arecoline in combination with various pathway inhibitors, and the expression of transforming growth factor-beta isoform genes and of collagen isoforms was assessed using reverse transcription polymerase chain reaction analysis. Results: We observed the induction of transforming growth factor-beta2 by arecoline in HaCaT cells and this induction was found to be caused by activation of the M-3 muscarinic acid receptor via the induction of calcium and the protein kinase C pathway. Most importantly, we showed that transforming growth factor-beta2 was significantly overexpressed in oral submucous fibrosis tissues (p = 0.008), with a median of 2.13 (n = 21) compared with 0.75 (n = 18) in normal buccal mucosal tissues. Furthermore, arecoline down-regulated the expression of collagens 1A1 and 3A1 in human primary gingival fibroblasts; however these collagens were induced by arecoline in the presence of spent medium of cultured human keratinocytes. Treatment with a transforming growth factor-beta blocker, transforming growth factor-beta1 latency-associated peptide, reversed this up-regulation of collagen, suggesting a role for profibrotic cytokines, such as transforming growth factor-beta, in the induction of collagens. Conclusion: Taken together, our data highlight the importance of arecoline-induced epithelial changes in the pathogenesis of oral submucous fibrosis.

Solution NMR studies of acetohydroxy acid synthase I: Identification of the sites of inter-subunit interactions using multidimensional NMR methods

The novel multidomain organization in the multimeric Escherichia coli AHAS I (ilvBN) enzyme has been dissected to generate polypeptide fragments. These fragments when cloned, expressed and purified reassemble in the presence of cofactors to yield a catalytically competent enzyme. Structural characterization of AHAS has been impeded due to the fact that the holoenzyme is prone to dissociation leading to heterogeneity in samples. Our approach has enabled the structural characterization using high-resolution nuclear magnetic resonance methods. Near complete sequence specific NMR assignments for backbone H-N, N-15, C-13 alpha and C-13(beta) atoms of the FAD binding domain of ilvB have been obtained on samples isotopically enriched in H-2, C-13 and N-15. The secondary structure determined on the basis of observed C-13(alpha) secondary chemical shifts and sequential NOEs indicates that the secondary structure of the FAD binding domain of E. coli AHAS large Subunit (ilvB) is similar to the structure of this domain in the catalytic subunit of yeast AHAS. Protein-protein interactions involving the regulatory subunit (ilvN) and the domains of the catalytic subunit (ilvB) were studied using circular dichroic and isotope edited solution nuclear magnetic resonance spectroscopic methods. Observed changes in circular dichroic spectra indicate that the regulatory subunit (ilvN) interacts with ilvB alpha and ilvB beta domains of the catalytic subunit and not with the ilvB gamma domain. NMR chemical shift mapping methods show that ilvN binds close to the FAD binding site in ilvB beta and proximal to the intrasubunit ilvB alpha/ilvB beta domain interface. The implication of this interaction on the role of the regulatory subunit oil the activity of the holoenzyme is discussed. NMR studies of the regulatory domains show that these domains are structured in solution. Preliminary evidence for the interaction of ilvN with the metabolic end product of the pathway, viz., valine is also presented.

Differentiation of neural stem cells in fragile X syndrome

Multipotent stem cells can self-renew and give rise to multiple cell types. One type of mammalian multipotent stem cells are neural stem cells (NSC)s, which can generate neurons, astrocytes and oligodendrocytes. NSCs are likely involved in learning and memory, but their exact role in cognitive function in the developing and adult brain is unclear. We have studied properties of NSCs in fragile X syndrome (FXS), which is the most common form of inherited mental retardation. FXS is caused by the lack of functional fragile X mental retardation protein (FMRP). FMRP is involved in the regulation of postsynaptic protein synthesis in a group I metabotropic glutamate receptor 5 (mGluR5)-dependent manner. In the absence of functional FMRP, the formation of functional synapses is impaired in the forebrain which results in alterations in synaptic plasticity. In our studies, we found that FMRP-deficient NSCs generated more neurons and less glia than control NSCs. The newborn neurons derived from FMRP-deficient NSCs showed an abnormally immature morphology. Furthermore, FMRP-deficient NSCs exhibited aberrant oscillatory Ca2+ responses to glutamate, which were specifically abolished by an antagonist of the mGluR5 receptor. The data suggested alterations in glutamatergic differentiation of FMRP-deficient NSCs and were further supported by an accumulation of cells committed to glutamatergic lineage in the subventricular zone of the embryonic Fmr1-knockout (Fmr1-KO) neocortex. Postnatally, the aberrant cells likely contributed to abnormal formation of the neocortex. The findings suggested a defect in the differentiation of distinct glutamatergic mGluR5 responsive cells in the absence of functional FMRP. Furthermore, we found that in the early postnatal Fmr1-KO mouse brain, the expression of mRNA for regulator of G-protein signalling-4 (RGS4) was decreased which was in line with disturbed G-protein signalling in NSCs lacking FMRP. Brain derived neurotrophic factor (BDNF) promotes neuronal differentiation of NSCs as the absence of FMRP was shown to do. This led us to study the effect of impaired BDNF/TrkB receptor signaling on NSCs by overexpression of TrkB.T1 receptor isoform. We showed that changes in the relative expression levels of the full-length and truncated TrkB isoforms influenced the replication capacity of NSCs. After the differentiation, the overexpression of TrkB.T1 increased neuronal turnover. To summarize, FMRP and TrkB signaling are involved in normal differentiation of NSCs in the developing brain. Since NSCs might have potential for therapeutic interventions in a variety of neurological disorders, our findings may be useful in the design of pharmacological interventions in neurological disorders of learning and memory.

Nuclear Import Mechanisms of STAT and NF-kB Transcription Factors

The eukaryotic cell nucleoplasm is separated from the cytoplasm by the nuclear envelope. This compartmentation of eukaryotic cells requires that all nuclear proteins must be transported from the cytoplasm into the nucleus. Transport of macromolecules between the nucleus and the cytoplasm occurs through nuclear pore complexes (NPCs). Proteins to be targeted into the nucleus by the classical nuclear import system contain nuclear localization signals (NLSs), which are recognized by importin alpha, the NLS receptor. Importin alpha binds to importin beta, which docks the importin-cargo complex on the cytoplasmic side of the NPC and mediates the movement of the complex into the nucleus. Presently six human importin alpha isoforms have been identified. Transcription factors are among the most important regulators of gene expression in eukaryotic organisms. Transcription factors bind to specific DNA sequences on target genes and modulate the activity of the target gene. Many transcription factors, including signal transducers and activators of transcription (STAT) and nuclear factor kB (NF-kB), reside in the cytoplasm in an inactive form, and upon activation they are rapidly transported into the nucleus. In the nucleus STATs and NF-kB regulate the activity of genes whose products are critical in controlling numerous cellular and organismal processes, such as inflammatory and immune responses, cell growth, differentiation and survival. The aim of this study was to investigate the nuclear import mechanisms of STAT and NF-kB transcription factors. This work shows that STAT1 homodimers and STAT1/STAT2 heterodimers bind specifically and directly to importin alpha5 molecule via unconventional dimer-specific NLSs. Importin alpha molecules have two regions, which have been shown to directly interact with the amino acids in the NLS of the cargo molecule. The Arm repeats 2-4 comprise the N-terminal NLS binding site and Arm repeats 7-8 the C-terminal NLS binding site. In this work it is shown that the binding site for STAT1 homodimers and STAT1/STAT2 heterodimers is composed of Arm repeats 8 and 9 of importin alpha5 molecule. This work demonstrates that all NF-kB proteins are transported into the nucleus by importin alpha molecules. In addition, NLS was identified in RelB protein. The interactions between NF-kB proteins and importin alpha molecules were found to be directly mediated by the NLSs of NF-kB proteins. Moreover, we found that p50 binds to the N-terminal and p65 to the C-terminal NLS binding site of importin alpha3. The results from this thesis work identify previously uncharacterized mechanisms in nuclear import of STAT and NF-kB. These findings provide new insights into the molecular mechanisms regulating the signalling cascades of these important transcription factors from the cytoplasm into the nucleus to the target genes.

Ligand dependent intra and inter subunit communication in human tryptophanyl tRNA synthetase as deduced from the dynamics of structure networks

Homodimeric protein tryptophanyl tRNA synthetase (TrpRS) has a Rossmann fold domain and belongs to the 1c subclass of aminoacyl tRNA synthetases. This enzyme performs the function of acylating the cognate tRNA. This process involves a number of molecules (2 protein subunits, 2 tRNAs and 2 activated Trps) and thus it is difficult to follow the complex steps in this process. Structures of human TrpRS complexed with certain ligands are available. Based on structural and biochemical data, mechanism of activation of Trp has been speculated. However, no structure has yet been solved in the presence of both the tRNA(Trp) and the activated Trp (TrpAMP). In this study, we have modeled the structure of human TrpRS bound to the activated ligand and the cognate tRNA. In addition, we have performed molecular dynamics (MD) simulations on these models as well as other complexes to capture the dynamical process of ligand induced conformational changes. We have analyzed both the local and global changes in the protein conformation from the protein structure network (PSN) of MD snapshots, by a method which was recently developed in our laboratory in the context of the functionally monomeric protein, methionyl tRNA synthetase. From these investigations, we obtain important information such as the ligand induced correlation between different residues of this protein, asymmetric binding of the ligands to the two subunits of the protein as seen in the crystal structure analysis, and the path of communication between the anticodon region and the aminoacylation site. Here we are able to elucidate the role of dimer interface at a level of detail, which has not been captured so far.

Pituitary gigantism treated successfully with the growth hormone receptor antagonist, pegvisomant

A Caucasian male aged 15 years presented with 2 years accelerated linear growth. He was 202 cm tall at presentation, with calculated mid-parental height of 173 cm. There were no features of hypopituitarism or acral growth. His visual fields and optic discs were normal...

Positional cloning and pathway analysis of the asthma susceptibility gene, NPSR1

In the present study, we identified a novel asthma susceptibility gene, NPSR1 (neuropeptide S receptor 1) on chromosome 7p14.3 by the positional cloning strategy. An earlier significant linkage mapping result among Finnish Kainuu asthma families was confirmed in two independent cohorts: in asthma families from Quebec, Canada and in allergy families from North Karelia, Finland. The linkage region was narrowed down to a 133-kb segment by a hierarchial genotyping method. The observed 77-kb haplotype block showed 7 haplotypes and a similar risk and nonrisk pattern in all three populations studied. All seven haplotypes occur in all three populations at frequences > 2%. Significant elevated relative risks were detected for elevated total IgE (immunoglobulin E) or asthma. Risk effects of the gene variants varied from 1.4 to 2.5. NPSR1 belongs to the G protein-coupled receptor (GPCR) family with a topology of seven transmembrane domains. NPSR1 has 9 exons, with the two main transcripts, A and B, encoding proteins of 371 and 377 amino acids, respectively. We detected a low but ubiquitous expression level of NPSR1-B in various tissues and endogenous cell lines while NPSR1-A has a more restricted expression pattern. Both isoforms were expressed in the lung epithelium. We observed aberrant expression levels of NPSR1-B in smooth muscle in asthmatic bronchi as compared to healthy. In an experimental mouse model, the induced lung inflammation resulted in elevated Npsr1 levels. Furthermore, we demonstrated that the activation of NPSR1 with its endogenous agonist, neuropeptide S (NPS), resulted in a significant inhibition of the growth of NPSR1-A overexpressing stable cell lines (NPSR1-A cells). To determine which target genes were regulated by the NPS-NPSR1 pathway, NPSR1-A cells were stimulated with NPS, and differentially expressed genes were identified using the Affymetrix HGU133Plus2 GeneChip. A total of 104 genes were found significantly up-regulated and 42 down-regulated 6 h after NPS administration. The up-regulated genes included many neuronal genes and some putative susceptibility genes for respiratory disorders. By Gene Ontology enrichment analysis, the biological process terms, cell proliferation, morphogenesis and immune response were among the most altered. The expression of four up-regulated genes, matrix metallopeptidase 10 (MMP10), INHBA (activin A), interleukin 8 (IL8) and EPH receptor A2 (EPHA2), were verified and confirmed by quantitative reverse-transcriptase-PCR. In conclusion, we identified a novel asthma susceptibility gene, NPSR1, on chromosome 7p14.3. NPS-NPSR1 represents a novel pathway that regulates cell proliferation and immune responses, and thus may have functional relevance in the pathogenesis of asthma.

Kinetic characterization of rat brain type IIA sodium channel alpha-subunit stably expressed in a somatic cell line

1. The rat brain type IIA Na+ channel alpha-subunit was stably expressed in Chinese hamster ovary (CHO) cells. Current through the expressed Na+ channels was studied using the whole-cell configuration of the patch clamp technique. The transient Na+ current was sensitive to TTX and showed a bell-shaped peak current vs. membrane potential relation. 2. Na+ current inactivation was better described by the sum of two exponentials in the potential range -30 to +40 mV, with. a dominating fast component and a small slower component. 3. The steady-state inactivation, h(infinity), was related to potential by a Boltzmann distribution, underlying thr ee states of the inactivation gate. 4. Recovery of the channels from inactivation at different potentials in the range -70 to -120 mV were characterized by al? initial delay which decreased with hyperpolarization. The time course was well fitted by the sum of two exponentials. In this case the slower exponential was the major component, and both time constants decreased with hyperpolarization. 5. For a working description of the Na+ channel inactivation in this preparation, with a minimal deviation from the Hodgkin-Huxley model, a three-state scheme of the form O reversible arrow I-1 reversible arrow I-2 was proposed, replacing the original two-state scheme of the Hodgkin-Huxley model, and the rate constants are reported. 6. The instantaneous current-voltage relationship showed marked deviation from linearity and was satisfactorily fitted by the constant-field equation. 7. The time course of activation was described by an m(x) model. However, the best-fitted value of x varied with the membrane potential and had a mean value of 2. 8. Effective gating charge was determined to be 4.7e from the slope of the activation plot, plotted on a logarithmic scale. 9. The rate constants of activation, alpha(m) and beta(m), were determined. Their functional dependence on the membrane potential was investigated.

Receptor guanylyl cyclase C (GC-C): regulation and signal transduction

Receptor guanylyl cyclase C (GC-C) is the target for the gastrointestinal hormones, guanylin, and uroguanylin as well as the bacterial heat-stable enterotoxins. The major site of expression of GC-C is in the gastrointestinal tract, although this receptor and its ligands play a role in ion secretion in other tissues as well. GC-C shares the domain organization seen in other members of the family of receptor guanylyl cyclases, though subtle differences highlight some of the unique features of GC-C. Gene knock outs in mice for GC-C or its ligands do not lead to embryonic lethality, but modulate responses of these mice to stable toxin peptides, dietary intake of salts, and development and differentiation of intestinal cells. It is clear that there is much to learn in future about the role of this evolutionarily conserved receptor, and its properties in intestinal and extra-intestinal tissues.