Intestinal epithelial CD98: an oligomeric and multifunctional protein

The intestinal epithelial cell-surface molecule, CD98 is a type II membrane glycoprotein. Molecular orientation studies have demonstrated that the C-terminal tail of human CD98 (hCD98), which contains a PDZ-binding domain, is extracellular. In intestinal epithelial cells, CD98 is covalently linked to an amino-acid transporter with which it forms a heterodimer. This heterodimer associates with beta(1)-integrin and intercellular adhesion molecular 1 (ICAM-1) to form a macromolecular complex in the basolateral membranes of polarized intestinal epithelial cells. This review focuses on the multifunctional roles of CD98, including involvement in extracellular signaling, adhesion/polarity, and amino-acid transporter expression in intestinal epithelia. A role for CD98 in intestinal inflammation, such as Intestinal Bowel Disease (IBD), is also proposed.     

Role of Wnts in prostate cancer bone metastases

Prostate cancer (CaP) is unique among all cancers in that when it metastasizes to bone, it typically forms osteoblastic lesions (characterized by increased bone production). CaP cells produce many factors, including Wnts that are implicated in tumor-induced osteoblastic activity. In this prospectus, we describe our research on Wnt and the CaP bone phenotype. Wnts are cysteine-rich glycoproteins that mediate bone development in the embryo and promote bone production in the adult. Wnts have been shown to have autocrine tumor effects, such as enhancing proliferation and protecting against apoptosis. In addition, we have recently identified that CaP-produced Wnts act in a paracrine fashion to induce osteoblastic activity in CaP bone metastases. In addition to Wnts, CaP cells express the soluble Wnt inhibitor dickkopf-1 (DKK-1). It appears that DKK-1 production occurs early in the development of skeletal metastases, which results in masking of osteogenic Wnts, thus favoring osteolysis at the metastatic site. As metastases progress, DKK-1 expression decreases allowing for unmasking of Wnt's osteoblastic activity and ultimately resulting in osteosclerosis at the metastatic site. We believe that DKK-1 is one of the switches that transitions the CaP bone metastasis activity from osteolytic to osteoblastic. Wnt/DKK-1 activity fits a model of CaP-induced bone remodeling occurring in a continuum composed of an osteolytic phase, mediated by receptor activator of NFkB ligand (RANKL), parathyroid hormone-related protein (PTHRP) and DKK-1; a transitional phase, where environmental alterations promote expression of osteoblastic factors (Wnts) and decreases osteolytic factors (i.e., DKK-1); and an osteoblastic phase, in which tumor growth-associated hypoxia results in production of vascular endothelial growth factor and endothelin-1, which have osteoblastic activity. This model suggests that targeting both osteolytic activity and osteoblastic activity will provide efficacy for therapy of CaP bone metastases.     

The Herpes Simplex Virus Type 1 UL20 Protein and the Amino Terminus of Glycoprotein K (gK) Physically Interact with gB

Herpes simplex virus type 1 (HSV-1) glycoprotein K (gK) and the UL20 protein (UL20p) are strictly required for virus-induced cell fusion, and mutations within either the gK or UL20 gene cause extensive cell fusion (syncytium formation). We have shown that gK forms a functional protein complex with UL20p, which is required for all gK and UL20p-associated functions in the HSV-1 life cycle. Recently, we showed that the amino-terminal 82 amino acids (aa) of gK (gKa) were required for the expression of the syncytial phenotype of the mutant virus gBΔ28 lacking the carboxyl-terminal 28 amino acids of gB (V. N. Chouljenko, A. V. Iyer, S. Chowdhury, D. V. Chouljenko, and K. G. Kousoulas, J. Virol. 83:12301-12313, 2009). This work suggested that the amino terminus of gK may directly or indirectly interact with gB and/or other viral glycoproteins. Two-way coimmunoprecipitation experiments revealed that UL20p interacted with gB in infected cells. Furthermore, the gKa peptide was coimmunoprecipitated with gB but not gD. Three recombinant baculoviruses were constructed, expressing the amino-terminal 82 aa of gKa together with either the extracellular portion of gB (30 to 748 aa), gD (1 to 340 aa), or gH (1 to 792 aa), respectively. Coimmunoprecipitation experiments revealed that gKa physically interacted with the extracellular portions of gB and gH but not gD. Three additional recombinant baculoviruses expressing gKa and truncated gBs encompassing aa 30 to 154, 30 to 364, and 30 to 500 were constructed. Coimmunoprecipitation experiments showed that gKa physically interacted with all three truncated gBs. Computer-assisted prediction of possible gKa binding sites on gB suggested that gKa may interact predominantly with gB domain I (E. E. Heldwein, H. Lou, F. C. Bender, G. H. Cohen, R. J. Eisenberg, and S. C. Harrison, Science 313:217-220, 2006). These results imply that the gK/UL20p protein complex modulates the fusogenic properties of gB and gH via direct physical interactions.Herpes simplex virus type 1 (HSV-1) can enter into cells via the fusion of its viral envelope with cellular membranes. Also, the virus can spread from infected to uninfected cells by causing virus-induced cell fusion, allowing virions to enter into uninfected cells without being exposed to extracellular spaces. These membrane fusion phenomena are known to be mediated by viral glycoproteins and other viral proteins (reviewed in reference 36). Although wild-type viruses cause a limited amount of virus-induced cell fusion, certain mutations cause extensive virus-induced cell-to-cell fusion (syncytial, or syn, mutations). These syncytial mutations are located predominantly within the UL20 gene (5, 27, 28); the UL24 gene (25, 38); the UL27 gene, encoding glycoprotein gB (7, 15, 18, 32); and the UL53 gene, coding for gK (6, 11, 24, 34, 35, 37).The presence of syncytial mutations within different viral genes, as well as other accumulating evidence, suggests that virus-induced cell fusion is mediated by the concerted action and interactions of the viral glycoproteins gD, gB, and gH/gL as well as gK and the membrane protein UL20p. Specifically, recent studies have shown that gD interacts with both gB and gH/gL (1, 2, 21). However, gB and gH/gL can also interact with each other even in the absence of gD (3). In this membrane fusion model, the binding of gD to its cognate receptors, including nectin-1, herpesvirus entry mediator (HVEM), and other receptors (8, 19, 30, 39-42), is thought to trigger sequential conformational changes in gH/gL and gB causing the fusion of the viral envelope with cellular membranes during virus entry as well as fusion among cellular membranes (22, 23). The transient coexpression of gB, gD, and gH/gL causes cell-to-cell fusion (31, 43), suggesting that these four viral glycoproteins are necessary and sufficient for membrane fusion. However, this transient fusion system does not accurately depict virus-induced cell fusion. Specifically, viral glycoprotein K (gK) and the UL20 membrane protein (UL20p) have been shown to be strictly required for virus-induced cell fusion (10, 27, 29). Moreover, syncytial mutations within gK (6, 11, 24, 34, 35, 37) or UL20 (5, 27, 28) promote extensive virus-induced cell fusion, and viruses lacking gK enter more slowly than the wild-type virus into susceptible cells (17). In contrast, the transient coexpression of gK carrying a syncytial mutation with gB, gD, and gH/gL did not enhance cell fusion, while the coexpression of wild-type gK with gB, gD, and gH/gL was reported previously to inhibit cell fusion in certain cell lines (4). To date, there is no direct evidence that either gK or UL20p interacts with gB, gD, gH, or gL.The X-ray structure of the ectodomain of HSV-1 gB has been determined and was predicted to assume at least two major conformations, one of which may be necessary for the fusogenic properties of gB (23). Single-amino-acid changes within the carboxyl terminus of gB located intracellularly as well as the deletion of the terminal 28 amino acids (aa) of gB cause extensive virus-induced cell fusion, presumably because they alter the extracellular conformation of gB (15, 31, 43). We have previously shown that HSV-1 gK and UL20p functionally and physically interact and that these interactions are absolutely necessary for their coordinate intracellular transport, cell surface expression, and functions in the HSV-1 life cycle (13, 16). In contrast to gB, syncytial mutations in gK map predominantly within extracellular domains of gK and particularly within the amino-terminal portion of gK (domain I) (12), while syncytial mutations of UL20 are located within the amino terminus of UL20p shown to be located intracellularly (27).Recently, we showed that the a peptide composed of the amino-terminal 82 amino acids of gK (gKa) can complement in trans for gB-mediated cell fusion caused by the deletion of the carboxyl-terminal 28 amino acids of gB, suggesting that the gKa peptide interacted with gB or other viral glycoproteins involved in virus-induced cell fusion (10). In this work, we demonstrate that UL20p and the amino terminus of gKa physically interact with gB in infected cells, while the gKa peptide is also capable of binding to the extracellular portion of gH, suggesting that gK/UL20p modulates virus-induced cell fusion via direct interactions with gB and gH.     

Arabidopsis extra-large G proteins (XLGs) regulate root morphogenesis

As in mammalian systems, heterotrimeric G proteins, composed of alpha, beta and gamma subunits, are present in plants and are involved in the regulation of development and cell signaling. Besides the sole prototypical G protein alpha subunit gene, GPA1, the Arabidopsis thaliana genome has three extra-large GTP-binding protein (XLG)-encoding genes: XLG1 (At2g23460), XLG2 (At4g34390) and XLG3 (At1g31930). The C-termini of the XLGs are Galpha domains that are homologous to GPA1, whereas their N-termini each contain a cysteine-rich region and a putative nuclear localization signal (NLS). GFP fusions with each XLG confirmed nuclear localization. All three XLG genes are expressed in essentially all plant organs, with strong expression in vascular tissues, primary root meristems and lateral root primordia. Analysis of single, double and triple T-DNA insertional mutants of the XLG genes revealed redundancy in XLG function. Dark-grown xlg1-1 xlg2-1 xlg3-1 triple mutant plants showed markedly increased primary root length compared with wild-type plants. This phenotype was not observed in dark-grown xlg single mutants, and was suppressed upon complementation of the xlg triple mutant with each XLG. Root cell sizes of the xlg triple mutant and root morphology were highly similar to those of wild-type roots, suggesting that XLGs may regulate cell proliferation. Dark-grown roots of the xlg triple mutants also showed altered sensitivity to sugars, ABA hyposensitivity and ethylene hypersensitivity, whereas seed germination in xlg triple mutants was hypersensitive to osmotic stress and ABA. As plant-specific proteins, regulatory mechanisms of XLGs may differ from those of conventional Galphas.     

Protein 4.1N does not interact with the inositol 1,4,5-trisphosphate receptor in an epithelial cell line

Cytosolic Ca2+ regulates a variety of cell functions, and the spatial patterns of Ca2+ signals are responsible in part for the versatility of this second messenger. The subcellular distribution of the inositol 1,4,5-trisphosphate receptor (IP3R) is thought to regulate Ca2+-signaling patterns but little is known about how the distribution of the IP3R itself is regulated. Here we examined the relationship between the IP3R and the cytoskeletal linker protein 4.1N in the polarized WIF-B cell line because protein 4.1N regulates targeting of the type I IP3R in neurons, but WIF-B cells do not express this cytoskeletal protein. WIF-B cells expressed all three isoforms of the IP3R, and each isoform was distributed throughout the cell. These cells did not express the ryanodine receptor. Photorelease of microinjected, caged IP3 induced a rapid rise in cytosolic Ca2+, but the increase began uniformly throughout the cell rather than at a specific initiation site. Expression of protein 4.1N was not associated with redistribution of the IP3R or changes in Ca2+-signaling patterns. These findings are consistent with the hypothesis that the subcellular distribution of IP3R isoforms regulates the formation of Ca2+ waves, and the finding that interactions between protein 4.1N and the IP3R vary among cell types may provide an additional, tissue-specific mechanism to shape the pattern of Ca2+ waves.     

Habitat destruction in mutualistic metacommunities

We investigate a mutualistic metacommunity where the strength of the mutualistic interaction between species is measured by the extent to which the presence of one species on a patch either reduces the extinction rate of the others present on the same patch or increases their ability to colonize other patches. In both cases, a strong enough mutualism enables all species to persist at habitat densities where they would all be extinct in the absence of the interaction. However, a mutualistic interaction that enhances colonization enables the species to persist at lower habitat density than one that suppresses extinction. All species abruptly go extinct (catastrophe) when the habitat density is decreased infinitesimally below a critical value. A comparison of the mean field or spatially implicit case with unrestricted dispersal and colonization to all patches in the system with a spatially explicit case where dispersal is restricted to the immediate neighbours of the original patch leads to the intriguing conclusion that restricted dispersal can be favourable for species that have a beneficial effect on each other when habitat conditions are adverse. When the mutualistic interaction is strong enough, the extinction threshold or critical amount of habitat required for the persistence of all species is lower when the dispersal is locally restricted than when unrestricted ! The persistence advantage for all species created by the mutualistic interaction increases substantially with the number of species in the metacommunity, as does the advantage for restricted dispersal over global dispersal.     

The Arabidopsis putative G protein-coupled receptor GCR1 interacts with the G protein alpha subunit GPA1 and regulates abscisic acid signaling

Heterotrimeric G proteins composed of alpha, beta, and gamma subunits link ligand perception by G protein-coupled receptors (GPCRs) with downstream effectors, providing a ubiquitous signaling mechanism in eukaryotes. The Arabidopsis thaliana genome encodes single prototypical Galpha (GPA1) and Gbeta (AGB1) subunits, and two probable Ggamma subunits (AGG1 and AGG2). One Arabidopsis gene, GCR1, encodes a protein with significant sequence similarity to nonplant GPCRs and a predicted 7-transmembrane domain structure characteristic of GPCRs. However, whether GCR1 actually interacts with GPA1 was unknown. We demonstrate by in vitro pull-down assays, by yeast split-ubiquitin assays, and by coimmunoprecipitation from plant tissue that GCR1 and GPA1 are indeed physically coupled. GCR1-GPA1 interaction depends on intracellular domains of GCR1. gcr1 T-DNA insertional mutants exhibit hypersensitivity to abscisic acid (ABA) in assays of root growth, gene regulation, and stomatal response. gcr1 guard cells are also hypersensitive to the lipid metabolite, sphingosine-1-phosphate (S1P), which is a transducer of the ABA signal upstream of GPA1. Because gpa1 mutants exhibit insensitivity in aspects of guard cell ABA and S1P responses, whereas gcr1 mutants exhibit hypersensitivity, GCR1 may act as a negative regulator of GPA1-mediated ABA responses in guard cells.     

Effect of Dietary Incorporation of Dry-Powdered Water Hyacinth (<Emphasis Type="Italic">Eichhornia crassipes</Emphasis>) Meal on Growth and Digestibility of <Emphasis Type="Italic">Labeo rohita</Emphasis> Fingerlings

Keeping the importance and search for unconventional feed resources and/or standardizing their level of incorporation in mind, we incorporated dry-powdered water hyacinth (Eichhornia crassipes) meal in feeds and studied its effect on growth and digestibility in Labeo rohita fingerlings. Five feeds with 30 % crude protein level were formulated using Eichhornia meal (EM) at 0 (control), 5 (EMF1), 10 (EMF2), 15 (EMF3) or 20 % (EMF4) of the diet replacing rice bran by equal proportions. Three hundred fingerlings (7.40 ± 0.05 cm; 5.27 ± 0.12 g) were distributed into fifteen tanks (200 l capacity) and fed the experimental diets for 60 days. In the last 30 days, digestibility studies were conducted using 0.5 % chromic oxide as an external marker in feed. At 10 % inclusion of EM, the experimental fish showed the highest weight gain percent (WG%), specific growth rate (SGR), protein efficiency ratio and apparent net protein utilization with lowest feed conversion ratio. Whereas the growth performance at 15 % inclusion level was comparable with the control and further increase to 20 % level of EM showed reduced growth responses but the feed was fairly palatable to the fish. Lower digestibility was also observed in EMF4 group. It is concluded that EM can be included at 15 % level in the feed of L. rohita fingerlings without adversely affecting the growth, dry matter and nutrient digestibility. However, economic feasibility of this feedstuff needs to be analyzed to see whether the reduced cost of diets would compensate for the reduced performance of fish at higher inclusion levels.