Localization of β2-microglobulin in the term villous syncytiotrophoblast

The non-covalent association of beta 2-microglobulin with MHC class I molecules and MHC class I-type molecules such as FcRn or the hemochromatosis protein (HFE) is of major importance for their function, i.e., antigen presentation, IgG transport, and regulation of iron uptake, respectively. In the human hemochorial placenta, the syncytiotrophoblast forms a continuous epithelial layer covering the villous trees, where it directly contacts maternal blood and, among many other functions, mediates uptake of maternal IgG and iron. The villous syncytiotrophoblast lacks MHC class I molecules but expresses FcRn and HFE. Since data on beta 2-microglobulin synthesis and localization in the term villous syncytiotrophoblast were contradictory, we investigated the subcellular localization of beta 2-microglobulin by immunoelectron microscopy. Synthesis in the trophoblast is demonstrated by colocalization of beta 2-microglobulin with protein disulfide isomerase, a marker protein of the endoplasmic reticulum. The presence of beta 2-microglobulin at the apical plasma membrane corresponds to the recently observed association of beta 2-microglobulin with HFE and FcRn. Localization of beta 2-microglobulin in late endosomes/lysosomes, labeled with antibodies to lysosome membrane antigen LAMP 2, suggests also a degradative route of beta 2-microglobulin internalized by fluid-phase from the maternal blood.     

Surfactant Protein-A Modulates LPS-Induced TLR4 Localization and Signaling via β-Arrestin 2

The soluble C-type lectin surfactant protein (SP)-A mediates lung immune responses partially via its direct effects on alveolar macrophages (AM), the main resident leukocytes exposed to antigens. SP-A modulates the AM threshold of lipopolysaccharide (LPS) activity towards an anti-inflammatory phenotype both in vitro and in vivo through various mechanisms. LPS responses are tightly regulated via distinct pathways including subcellular TLR4 localization and thus ligand sensing. The cytosolic scaffold and signaling protein β-arrestin 2 acts as negative regulator of LPS-induced TLR4 activation. Here we show that SP-A neither increases TLR4 abundancy nor co-localizes with TLR4 in primary AM. SP-A significantly reduces the LPS-induced co-localization of TLR4 with the early endosome antigen (EEA) 1 by promoting the co-localization of TLR4 with the post-Golgi compartment marker Vti1b in freshly isolated AM from rats and wild-type (WT) mice, but not in β-arrestin 2^−/− AM. Compared to WT mice pulmonary LPS-induced TNF-α release in β-arrestin 2^−/− mice is accelerated and enhanced and exogenous SP-A fails to inhibit both lung LPS-induced TNF-α release and TLR4/EEA1 positioning. SP-A, but not LPS, enhances β-arrestin 2 protein expression in a time-dependent manner in primary rat AM. The constitutive expression of β-arrestin 2 in AM from SP-A^−/− mice is significantly reduced compared to SP-A^+/+ mice and is rescued by SP-A. Prolonged endosome retention of LPS-induced TLR4 in AM from SP-A^−/− mice is restored by exogenous SP-A, and is antagonized by β-arrestin 2 blocking peptides. LPS induces β-arrestin 2/TLR4 association in primary AM which is further enhanced by SP-A. The data demonstrate that SP-A modulates LPS-induced TLR4 trafficking and signaling in vitro and in vivo engaging β-arrestin 2.     

Recruitment of β-Arrestin into Neuronal Cilia Modulates Somatostatin Receptor Subtype 3 Ciliary Localization

Primary cilia are essential sensory and signaling organelles present on nearly every mammalian cell type. Defects in primary cilia underlie a class of human diseases collectively termed ciliopathies. Primary cilia are restricted subcellular compartments, and specialized mechanisms coordinate the localization of proteins to cilia. Moreover, trafficking of proteins into and out of cilia is required for proper ciliary function, and this process is disrupted in ciliopathies. The somatostatin receptor subtype 3 (Sstr3) is selectively targeted to primary cilia on neurons in the mammalian brain and is implicated in learning and memory. Here, we show that Sstr3 localization to cilia is dynamic and decreases in response to somatostatin treatment. We further show that somatostatin treatment stimulates β-arrestin recruitment into Sstr3-positive cilia and this recruitment can be blocked by mutations in Sstr3 that impact agonist binding or phosphorylation. Importantly, somatostatin treatment fails to decrease Sstr3 ciliary localization in neurons lacking β-arrestin 2. Together, our results implicate β-arrestin in the modulation of Sstr3 ciliary localization and further suggest a role for β-arrestin in the mediation of Sstr3 ciliary signaling.     

Localization of β-1,3-glucanase in Trichoderma harzianum

Studies on the constitutive β-1,3-glucanase were conducted in submerged as well as in the stationary culture conditions, in the presence and in the absence of lactose and glucose as main carbon sources. In the absence of lactose or glucose, expression of β-1,3-glucanase was observed at 96?h in extracellular, periplasmic, cell wall bound and internal fractions during submerged fermentation. In shake flask culture, enzyme was found in all subcellular fractions using optimal glucose concentration. When Trichoderma harzianum was grown on media containing 55?kg lactose/m³ in submerged culture, activity was found in extracellular, cell wall bound and in the periplasmic fractions. The relative distribution of the enzyme in the cell is independent of the nature of the carbon source and its concentration.     

Distinct Roles of β-Arrestin 1 and β-Arrestin 2 in ORG27569-induced Biased Signaling and Internalization of the Cannabinoid Receptor 1 (CB1)

The cannabinoid receptor 1 (CB1) is a G protein-coupled receptor primarily expressed in brain tissue that has been implicated in several disease states. CB1 allosteric compounds, such as {"type":"entrez-protein","attrs":{"text":"ORG27569","term_id":"1179174593","term_text":"ORG27569"}}ORG27569, offer enormous potential as drugs over orthosteric ligands, but their mechanistic, structural, and downstream effects upon receptor binding have not been established. Previously, we showed that {"type":"entrez-protein","attrs":{"text":"ORG27569","term_id":"1179174593","term_text":"ORG27569"}}ORG27569 enhances agonist binding affinity to CB1 but inhibits G protein-dependent agonist signaling efficacy in HEK293 cells and rat brain expressing the CB1 receptor (Ahn, K. H., Mahmoud, M. M., and Kendall, D. A. (2012) J. Biol. Chem. 287, 12070–12082). Here, we identify the mediators of CB1 receptor internalization and {"type":"entrez-protein","attrs":{"text":"ORG27569","term_id":"1179174593","term_text":"ORG27569"}}ORG27569-induced G protein-independent signaling. Using siRNA technology, we elucidate an {"type":"entrez-protein","attrs":{"text":"ORG27569","term_id":"1179174593","term_text":"ORG27569"}}ORG27569-induced signaling mechanism for CB1 wherein β-arrestin 1 mediates short term signaling to ERK1/2 with a peak at 5 min and other upstream kinase components including MEK1/2 and c-Src. Consistent with these findings, we demonstrate co-localization of CB1-GFP with red fluorescent protein-β-arrestin 1 upon {"type":"entrez-protein","attrs":{"text":"ORG27569","term_id":"1179174593","term_text":"ORG27569"}}ORG27569 treatment using confocal microscopy. In contrast, we show the critical role of β-arrestin 2 in CB1 receptor internalization upon treatment with CP55940 (agonist) or treatment with {"type":"entrez-protein","attrs":{"text":"ORG27569","term_id":"1179174593","term_text":"ORG27569"}}ORG27569. These results demonstrate for the first time the involvement of β-arrestin in CB1-biased signaling by a CB1 allosteric modulator and also define the differential role of the two β-arrestin isoforms in CB1 signaling and internalization.     

Neurite Outgrowth-stimulating Activities of β-Casomorphins in Neuro-2a Mouse Neuroblastoma Cells

Endogenous opioid peptides and opiate drugs are known to affect the development of the nervous system. β-Casomorphins (β-CMs) belong to a family of exogenous opioid peptides derived from the milk protein β-casein by proteolytic fragmentation. We investigated the effects of various fragments and analogues of β-CM on neurite outgrowth in Neuro-2a mouse neuroblastoma cells. The fragments β-CM-5 to -9 and β-CM-5 amide stimulated neurite outgrowth. Fragments shorter than β-CM-5 (β-CM-3, -4, and β-CM-4 amide) and longer than β-CM-9 (β-CM-13 and -21) had no effects. The activity of β-CMs to promote neurite outgrowth does not correlate with their opioid activity in guinea-pig ileum. The effect of the most potent fragment, β-CM-5, was prevented by the μ-opioid receptor-selectiveantagonist D-Phe-Cys²-Tyr³-D-Trp-Orn⁵-Thr⁶-Pen⁷- Thr⁸-NH₂ (CTOP), or by pretreatment with pertussis toxin. These results suggest that the stimulatory effects of β-CMs on neurite outgrowth were mediated through G protein-coupled μ-opioid receptors.     

Localization of β-glycerophosphatase activity in cotton fiber during differentiation

Summary The distribution of -glycerophosphatase activity in the outer integument of cotton (Gossypium hirsutum L.) ovules was determined histochemically at the electron microscope level. Both a linted cultivar and a lintless mutant line were examined from 1 day preanthesis to 3 days postanthesis. No enzyme activity was observed in the lintless line at any stage. In the linted cultivar there was no enzyme activity before anthesis, but as fibers were initiated on the day of anthesis, activity was observed in the expanding fiber cell wall and nucleus. As the fibers started elongating, enzyme activity was particularly concentrated in the cytoplasm and wall where directional growth towards the micropyle occurs. By 2 days postanthesis, -glycerophosphatase activity was decreasing in the cell wall and nucleus, but was increasing in the nucleolus. Enzyme activity in the nucleolus was highest at 3 days post-anthesis, but nuclear -glycerophosphatase activity was not observed then. These results indicate that -glycerophosphatase activity was associated with differentiating fiber cells, specifically with those sites where distinct anatomical, and perhaps catabolic, changes were occurring. The significance of the results is discussed in relation to possible mechanisms of cotton fiber differentiation.Abbreviations EM Electron microscopic - ER Endoplasmic reticulum - GP -Glycerophosphate - GPase -Glycerophosphatase - SEM Scanning electron microscopy     

Specific recognition in the tertiary structure of β-sheets of proteins

The frequency of occurrence of nearest neighbour residue pairs on adjacent antiparallel (β_A) and parallel (β_P) strands is obtained from 30 known protein structures. The specificity of interstrand recognition due to such pairing as a factor in the folding of β-sheets is studied by statistical methods. Residues of sufficiently high count for statistical analysis are treated individually while the rest are combined into small groups of similar size, polarity, and/or genetic exchangeability. The hypothesis of specific recognition between individuals and small groups is contrasted with the alternative hypothesis of non-specific recognition between broad classes (hydrophobia, neutral, polar) of residues. A χ² test of pair correlations favours specific recognition against non-specific recognition with a high level of confidence. The largest and most significant correlations are: Ser/Thr (1.9 ± 0.3), Ile/Val (1.7 ± 0.3) and Lys-Arg/Asp-Gln (1.8 ± 0.3) in β_A, and Ile/Leu (1.9 ± 0.4) in β_P. The pair Gly/Gly never occurs in any β-sheet. The specific residue-pair correlations derived here may be useful in statistical prediction methods of protein tertiary structure.     

Targeting of β-Arrestin2 to the Centrosome and Primary Cilium: Role in Cell Proliferation Control

Background

The primary cilium is a sensory organelle generated from the centrosome in quiescent cells and found at the surface of most cell types, from where it controls important physiological processes. Specific sets of membrane proteins involved in sensing the extracellular milieu are concentrated within cilia, including G protein coupled receptors (GPCRs). Most GPCRs are regulated by β-arrestins, βarr1 and βarr2, which control both their signalling and endocytosis, suggesting that βarrs may also function at primary cilium.

Methodology/Principal Findings

In cycling cells, βarr2 was observed at the centrosome, at the proximal region of the centrioles, in a microtubule independent manner. However, βarr2 did not appear to be involved in classical centrosome-associated functions. In quiescent cells, both in vitro and in vivo, βarr2 was found at the basal body and axoneme of primary cilia. Interestingly, βarr2 was found to interact and colocalize with 14-3-3 proteins and Kif3A, two proteins known to be involved in ciliogenesis and intraciliary transport. In addition, as suggested for other centrosome or cilia-associated proteins, βarrs appear to control cell cycle progression. Indeed, cells lacking βarr2 were unable to properly respond to serum starvation and formed less primary cilia in these conditions.

Conclusions/Significance

Our results show that βarr2 is localized to the centrosome in cycling cells and to the primary cilium in quiescent cells, a feature shared with other proteins known to be involved in ciliogenesis or primary cilium function. Within cilia, βarr2 may participate in the signaling of cilia-associated GPCRs and, therefore, in the sensory functions of this cell “antenna”.     

Identification of a Karyopherin β1/β2 Proline-Tyrosine Nuclear Localization Signal in Huntingtin Protein

Among the known pathways of protein nuclear import, the karyopherin β2/transportin pathway is only the second to have a defined nuclear localization signal (NLS) consensus. Huntingtin, a 350-kDa protein, has defined roles in the nucleus, as well as a CRM1/exportin-dependent nuclear export signal; however, the NLS and exact pathway of import have remained elusive. Here, using a live cell assay and affinity chromatography, we show that huntingtin has a karyopherin β2-dependent proline-tyrosine (PY)-NLS in the amino terminus of the protein. This NLS comprises three consensus components: a basic charged sequence, a downstream conserved arginine, and a PY sequence. Unlike the classic PY-NLS, which has an unstructured intervening sequence between the consensus components, we show that a β sheet structured region separating the consensus elements is critical for huntingtin NLS function. The huntingtin PY-NLS is also capable of import through the importin/karyopherin β1 pathway but was not functional in all cell types tested. We propose that this huntingtin PY-NLS may comprise a new class of multiple import factor-dependent NLSs with an internal structural component that may regulate NLS activity.     

Localization of β-oxidation enzymes in peroxisomes isolated from nonfatty plant tissues

Peroxisomes from spinach leaves, mungbean hypocotyls, and potato tubers catalyze a palmitoyl-CoA-dependent, KCN-insensitive O₂ uptake. In the course of this reaction O₂ is reduced to H₂O₂ in a 1:1 stoichiometry and palmitoyl-CoA oxidized, in a 1:1 stoichiometry, to a product serving as substrate for enoyl-CoA hydratase. These findings demonstrate the existence of a peroxisomal acyl-CoA oxidase in these tissues. Enoyl-CoA hydratase (EC 4.2.1.17), 3-hydroxyacyl-CoA dehydrogenase (EC 1.1.1.35), and thiolase (EC 2.3.1.9) are also associated with the peroxisomes from mung-bean hypocotyls and potato tubers (as well as with spinach leaf peroxisomes as recently reported; Gerhardt 1981, FEBS Lett. 126, 71). The low activities of these enzymes in mitochondrial fractions seem to be due to contaminating peroxisomes since the ratio of β-oxidation enzyme activities to catalase activity did not significantly differ between peroxisomal and mitochondrial fractions isolated on sucrose density gradients. The proof of localization of β-oxidation enzymes in peroxisomes without glyoxysomal function leads to the concept that fatty-acid oxidation is a consistent basic function of the peroxisome in cells of higher plants.     

Localization of β1-Adrenergic Receptors in the Cochlea and the Vestibular Labyrinth

Sympathetic activation in a “fight or flight reaction” may put the sensory systems for hearing and balance into a state of heightened alert via β₁-adrenergic receptors (β₁-AR). The aim of the present study was to localize β₁-AR in the gerbil inner ear by confocal immunocytochemistry, to characterize β₁-AR by Western immunoblots, and to identify β₁-AR pharmacologically by measurements of cAMP production. Staining for β₁-AR was found in strial marginal cells, inner and outer hair cells, outer sulcus, and spiral ganglia cells of the cochlea, as well as in dark, transitional and supporting cells of the vestibular labyrinth. Receptors were characterized in microdissected inner ear tissue fractions as 55 kDa non-glycosylated species and as 160 kDa high-mannose-glycosylated complexes. Pharmacological studies using isoproterenol, ICI-118551 and CGP-20712A demonstrated β₁-AR as the predominant adrenergic receptor in stria vascularis and organ of Corti. In conclusion, β₁-AR are present and functional in inner ear epithelial cells that are involved in K⁺ cycling and auditory transduction, as well as in neuronal cells that are involved in auditory transmission.     

Localization of luteinizing hormone β-mRNA by in situ hybridization in the sheep pars tuberalis

Summary The localization of luteinizing hormone beta (LH)-mRNA was studied by in situ hybridization in the pars tuberalis of sheep using a homologous sheep double-stranded ³²P-or ³⁵S-cDNA. The labelled cDNA probe detected one mRNA sequence in the pars tuberalis by Northern blot analysis; this sequence was similar to that detected in the pituitary. In situ, the labelling of LH-mRNA in the horizontal and sagittal tissue sections was found throughout the pars tuberalis. This labelling was prevented by adding an excess of cold probe or treating the sections by ribonuclease before in situ hybridization. Controls showed a labelling in the pars distalis, but not in the median eminence, hypothalamus, cerebral cortex and liver sections. Double labelling by using a specific LH-antiserum indicated that the labelling of LH-mRNA appeared more intense in LH-containing cells that were found only in the ventral part of the pars tuberalis. These results suggest that the entire pars tuberalis is able to produce the LH subunit, but that the level of translation greatly varies according to the location of the cells.     

Different Roles of G Protein Subunits β1 and β2 in Neutrophil Function Revealed by Gene Expression Silencing in Primary Mouse Neutrophils

Neutrophils play important roles in host innate immunity and various inflammation-related diseases. In addition, neutrophils represent an excellent system for studying directional cell migration. However, neutrophils are terminally differentiated cells that are short lived and refractory to transfection; thus, they are not amenable for existing gene silencing techniques. Here we describe the development of a method to silence gene expression efficiently in primary mouse neutrophils. A mouse stem cell virus-based retroviral vector was modified to express short hairpin RNAs and fluorescent marker protein at high levels in hematopoietic cells and used to infect mouse bone marrow cells prior to reconstitution of the hematopoietic system in lethally irradiated mice. This method was used successfully to silence the expression of Gβ₁ and/or Gβ₂ in mouse neutrophils. Knockdown of Gβ₂ appeared to affect primarily the directionality of neutrophil chemotaxis rather than motility, whereas knockdown of Gβ₁ had no significant effect. However, knockdown of both Gβ₁ and Gβ₂ led to significant reduction in motility and responsiveness. In addition, knockdown of Gβ₁ but not Gβ₂ inhibited the ability of neutrophils to kill ingested bacteria, and only double knockdown resulted in significant reduction in bacterial phagocytosis. Therefore, we have developed a short hairpin RNA-based method to effectively silence gene expression in mouse neutrophils for the first time, which allowed us to uncover divergent roles of Gβ₁ and Gβ₂ in the regulation of neutrophil functions.     

Gastrointestinal Hyperplasia with Altered Expression of DNA Polymerase β

Background

Altered expression of DNA polymerase β (Pol β) has been documented in a large percentage of human tumors. However, tumor prevalence or predisposition resulting from Pol β over-expression has not yet been evaluated in a mouse model.

Methodology/Principal Findings

We have recently developed a novel transgenic mouse model that over-expresses Pol β. These mice present with an elevated incidence of spontaneous histologic lesions, including cataracts, hyperplasia of Brunner''s gland and mucosal hyperplasia in the duodenum. In addition, osteogenic tumors in mice tails, such as osteoma and osteosarcoma were detected. This is the first report of elevated tumor incidence in a mouse model of Pol β over-expression. These findings prompted an evaluation of human gastrointestinal tumors with regard to Pol β expression. We observed elevated expression of Pol β in stomach adenomas and thyroid follicular carcinomas, but reduced Pol β expression in esophageal adenocarcinomas and squamous carcinomas.

Conclusions/Significance

These data support the hypothesis that balanced and proficient base excision repair protein expression and base excision repair capacity is required for genome stability and protection from hyperplasia and tumor formation.     

SUMOylation Attenuates Human β-Arrestin 2 Inhibition of IL-1R/TRAF6 Signaling

β-Arrestin 2 as an adaptor plays a role in the regulation of receptor desensitization, trafficking, and signaling. Bovine β-arrestin 2 has been shown to be SUMOylated on the lysine 400 residue, which links it to the endocytosis of the β₂-adrenergic receptor. Here we identify a major SUMOylation site, lysine 295, on human β-arrestin 2. SUMOylation on this site attenuates β-arrestin 2 binding to TRAF6, then enhances TRAF6 oligomerization and autoubiquitination, and consequently leads to the increase of TRAF6-mediated NF-κB/AP-1 activation. We further determine SENP1 as a specific de-SUMOylation protease that can reverse the SUMOylation of β-arrestin 2-mediated processes. Our study reveals SUMOylation as a novel mechanism in the regulation of β-arrestin 2-mediated IL-1R/TRAF6 signaling.     

Specific characteristics of CK2β regulatory subunits in plants

In all eukaryotes, the typical CK2 holoenzyme is an heterotetramer composed of two catalytic (CK2α and CK2α') and two regulatory (CK2β) subunits. One of the distinctive traits of plant CK2 is that they present a greater number of genes encoding for CK2α/β subunits than animals or yeasts, for instance, in Arabidopsis and maize both CK2α/β subunits belong to multigenic families composed by up to four genes. Here, we conducted a genome-wide survey examining 34 different plant genomes in order to investigate if the multigenic property of CK2β genes is a common feature through the entire plant kingdom. Also, at the level of structure, the plant CK2β regulatory subunits present distinctive features as (i) they lack about 20 aminoacids in the C-terminal domain, (ii) they present a specific N-terminal extension of about 90 aminoacids that shares no homology with any previously characterized functional domain, and (iii) the acidic loop region is poorly conserved at the aminoacid level. Since there is no data about CK2β or holoenzyme structure in plants, in this study, we use human CK2β as a template to predict a structure for Zea mays CK2β1 by homology modeling and we discuss about possible structural changes in the acidic loop region that could affect the enzyme regulation.