Membrane Mucin Muc4 promotes blood cell association with tumor cells and mediates efficient metastasis in a mouse model of breast cancer.

Mucin-4 (Muc4) is a large cell surface glycoprotein implicated in the protection and lubrication of epithelial structures. Previous studies suggest that aberrantly expressed Muc4 can influence the adhesiveness, proliferation, viability and invasiveness of cultured tumor cells, as well as the growth rate and metastatic efficiency of xenografted tumors. Although it has been suggested that one of the major mechanisms by which Muc4 potentiates tumor progression is via its engagement of the ErbB2/HER2 receptor tyrosine kinase, other mechanisms exist and remain to be delineated. Moreover, the requirement for endogenous Muc4 for tumor growth progression has not been previously explored in the context of gene ablation. To assess the contribution of endogenous Muc4 to mammary tumor growth properties, we first created a genetically engineered mouse line lacking functional Muc4 (Muc4ko), and then crossed these animals with the NDL (Neu DeLetion mutant) model of ErbB2-induced mammary tumorigenesis. We observed that Muc4ko animals are fertile and develop normally, and adult mice exhibit no overt tissue abnormalities. In tumor studies, we observed that although some markers of tumor growth such as vascularity and cyclin D1 expression are suppressed, primary mammary tumors from Muc4ko/NDL female mice exhibit similar latencies and growth rates as Muc4wt/NDL animals. However, the presence of lung metastases is markedly suppressed in Muc4ko/NDL mice. Interestingly, histological analysis of lung lesions from Muc4ko/NDL mice revealed a reduced association of disseminated cells with platelets and white blood cells. Moreover, isolated cells derived from Muc4ko/NDL tumors interact with fewer blood cells when injected directly into the vasculature or diluted into blood from wild type mice. We further observed that blood cells more efficiently promote the viability of non-adherent Muc4wt/NDL cells than Muc4ko/NDL cells. Together, our observations suggest that Muc4 may facilitate metastasis by promoting the association of circulating tumor cells with blood cells to augment tumor cell survival in circulation.

Suppression of planar cell polarity signaling and migration in glioblastoma by Nrdp1-mediated Dvl polyubiquitination.

The lethality of the aggressive brain tumor glioblastoma multiforme (GBM) results in part from its strong propensity to invade surrounding normal brain tissue. Although oncogenic drivers such as epidermal growth factor receptor activation and Phosphatase and Tensin homolog inactivation are thought to promote the motility and invasiveness of GBM cells via phosphatidylinostitol 3-kinase activation, other unexplored mechanisms may also contribute to malignancy. Here we demonstrate that several components of the planar cell polarity (PCP) arm of non-canonical Wnt signaling including VANGL1, VANGL2 and FZD7 are transcriptionally upregulated in glioma and correlate with poorer patient outcome. Knockdown of the core PCP pathway component VANGL1 suppresses the motility of GBM cell lines, pointing to an important mechanistic role for this pathway in glioblastoma malignancy. We further observe that restoration of Nrdp1, a RING finger type E3 ubiquitin ligase whose suppression in GBM also correlates with poor prognosis, reduces GBM cell migration and invasiveness by suppressing PCP signaling. Our observations indicate that Nrdp1 physically interacts with the Vangl1 and Vangl2 proteins to mediate the K63-linked polyubiquitination of the Dishevelled, Egl-10 and Pleckstrin (DEP) domain of the Wnt pathway protein Dishevelled (Dvl). Ubiquitination hinders Dvl binding to phosphatidic acid, an interaction necessary for efficient Dvl recruitment to the plasma membrane upon Wnt stimulation of Fzd receptor and for the propagation of downstream signals. We conclude that the PCP pathway contributes significantly to the motility and hence the invasiveness of GBM cells, and that Nrdp1 acts as a negative regulator of PCP signaling by inhibiting Dvl through a novel polyubiquitination mechanism. We propose that the upregulation of core PCP components, together with the loss of the key negative regulator Nrdp1, act coordinately to promote GBM invasiveness and malignancy.

Neoplastic transformation of porcine mammary epithelial cells in vitro and tumor formation in vivo.

BACKGROUND: The mammary glands of pigs share many functional and morphological similarities with the breasts of humans, raising the potential of their utility for research into the mechanisms underlying normal mammary function and breast carcinogenesis. Here we sought to establish a model for the efficient manipulation and transformation of porcine mammary epithelial cells (pMEC) in vitro and tumor growth in vivo. METHODS: We utilized a vector encoding the red florescent protein tdTomato to transduce populations of pMEC from Yorkshire -Hampshire crossbred female pigs in vitro and in vivo. Populations of primary pMEC were then separated by FACS using markers to distinguish epithelial cells (CD140a-) from stromal cells (CD140a+), with or without further enrichment for basal and luminal progenitor cells (CD49f+). These separated pMEC populations were transduced by lentivirus encoding murine polyomavirus T antigens (Tag) and tdTomato and engrafted to orthotopic or ectopic sites in immunodeficient NOD.Cg-Prkdc (scid) Il2rg (tm1Wjl) /SzJ (NSG) mice. RESULTS: We demonstrated that lentivirus effectively transduces pMEC in vitro and in vivo. We further established that lentivirus can be used for oncogenic-transformation of pMEC ex vivo for generating mammary tumors in vivo. Oncogenic transformation was confirmed in vitro by anchorage-independent growth, increased cell proliferation, and expression of CDKN2A, cyclin A2 and p53 alongside decreased phosphorylation of Rb. Moreover, Tag-transformed CD140a- and CD140a-CD49f + pMECs developed site-specific tumors of differing histopathologies in vivo. CONCLUSIONS: Herein we establish a model for the transduction and oncogenic transformation of pMEC. This is the first report describing a porcine model of mammary epithelial cell tumorigenesis that can be applied to the study of human breast cancers.

Cholesterol-dependent interaction of syncollin with the membrane of the pancreatic zymogen granule.

Syncollin is a protein of the pancreatic zymogen granule that was isolated through its ability to bind to syntaxin. Despite this in vitro interaction, it is now clear that syncollin is present on the luminal side of the zymogen granule membrane. Here we show that there are two pools of syncollin within the zymogen granule: one free in the lumen and the other tightly associated with the granule membrane. When unheated or cross-linked samples of membrane-derived syncollin are analysed by SDS/PAGE, higher-order forms are seen in addition to the monomer, which has an apparent molecular mass of 16 kDa. Extraction of cholesterol from the granule membrane by treatment with methyl-beta-cyclodextrin causes the detachment of syncollin, and this effect is enhanced at a high salt concentration. Purified syncollin is able to bind to brain liposomes at pH 5.0, but not at pH 11.0, a condition that also causes its extraction from granule membranes. Syncollin binds only poorly to dioleoyl phosphatidylcholine liposomes, but binding is dramatically enhanced by the inclusion of cholesterol. Finally, cholesterol can be co-immunoprecipitated with syncollin. We conclude that syncollin is able to interact directly with membrane lipids, and to insert into the granule membrane in a cholesterol-dependent manner. Membrane-associated syncollin apparently exists as a homo-oligomer, possibly consisting of six subunits, and its association with the membrane may be stabilized by electrostatic interactions with either other proteins or phospholipids.

ARF binds the C-terminal region of the Escherichia coli heat-labile toxin (LTA1) and competes for the binding of LTA2.

Cholera toxin (CT) and the heat-labile enterotoxin (LT) from Escherichia coli are highly related in terms of structure and biochemical activities and are the causative agents of cholera and traveler's diarrhea, respectively. The pathophysiological action of these toxins requires their activity as ADP-ribosyltransferases, transferring the ADP-ribose moiety from NAD onto the stimulatory, regulatory component of adenylyl cyclase, Gs. This reaction is highly dependent on the protein cofactor, termed ADP-ribosylation factor (ARF), that is itself a 20 kDa regulatory GTPase. In this study, we define sites of interaction between LTA and human ARF3. The residues identified as important to ARF binding include several of those previously shown to bind to the A2 subunit of the toxin and those important to the organization of two flexible loops, previously implicated as regulators of substrate entry. A model for how ARF acts to enhance the catalytic activity is proposed. A critical portion of the overlap between ARF and LTA(2) in binding LTA(1) includes a short region of sequence homology between LTA(2) and the switch II region of ARF. LTA(2) also interacted with ARF effectors in two-hybrid assays, and thus, we discuss the possibility that the LTA(2) subunit may function in cells as a partial ARF mimetic to compete for the binding of ARF to LTA(1) or regulate aspects of the toxin's transport from the cell surface to the ER.

Effects of glucocorticoids on adrenal chromaffin cells.

The mammalian adrenal gland consists of two anatomically distinct parts: an outer cortex that synthesizes steroids and a central medulla that contains catecholamine-producing chromaffin cells. Although derived from different embryological origins, the two secretory tissues in the adult animal are functionally as well as structurally linked. Glucocorticoids, a class of steroid hormones produced by the cortex, exert a variety of effects on medullary chromaffin cells. They modulate the expression of specific genes via activation of glucocorticoid receptors that act as transcription factors and either up- or down-regulate mRNA synthesis. The direct binding to and modulation of cation channels by glucocorticoids as well as the control of mRNA or protein stability are other proposed mechanisms of glucocorticoid action. The activity of phenylethanolamine N-methyltransferase, the enzyme that converts noradrenaline into adrenaline, is stimulated by glucocorticoids, which causes the conversion of noradrenergic to adrenergic chromaffin cells. Other phenotypic manifestations of glucocorticoid action include the upregulation of catecholamine synthesis, storage, and secretion. Furthermore, glucocorticoids have been implicated in chromaffin cell differentiation. However, recent gene knockout experiments suggest that glucocorticoid signalling is required only for the acquisition of the adrenergic but not the noradrenergic phenotype.

Dissection of a nuclear localization signal.

The regulated process of protein import into the nucleus of a eukaryotic cell is mediated by specific nuclear localization signals (NLSs) that are recognized by protein import receptors. This study seeks to decipher the energetic details of NLS recognition by the receptor importin alpha through quantitative analysis of variant NLSs. The relative importance of each residue in two monopartite NLS sequences was determined using an alanine scanning approach. These measurements yield an energetic definition of a monopartite NLS sequence where a required lysine residue is followed by two other basic residues in the sequence K(K/R)X(K/R). In addition, the energetic contributions of the second basic cluster in a bipartite NLS ( approximately 3 kcal/mol) as well as the energy of inhibition of the importin alpha importin beta-binding domain ( approximately 3 kcal/mol) were also measured. These data allow the generation of an energetic scale of nuclear localization sequences based on a peptide's affinity for the importin alpha-importin beta complex. On this scale, a functional NLS has a binding constant of approximately 10 nm, whereas a nonfunctional NLS has a 100-fold weaker affinity of 1 microm. Further correlation between the current in vitro data and in vivo function will provide the foundation for a comprehensive quantitative model of protein import.

Structural requirements for the specific recognition of an m7G mRNA cap.

7-Methylguanosine (m(7)G), also known as the mRNA "cap", is used as a molecular tag in eukaryotic cells to mark the 5' end of messenger RNAs. The mRNA cap is required for several key events in gene expression in which the m(7)G moiety is specifically recognized by cellular proteins. The configurations of the m(7)G-binding pockets of a cellular (eIF4E) and a viral (VP39) cap-binding protein have been determined by X-ray crystallography. The binding energy has been hypothesized to result from a pi-pi stacking interaction between aromatic residues sandwiching the m(7)G base in addition to hydrogen bonds between the base and acidic protein side chains. To further understand the structural requirements for the specific recognition of an m(7)G mRNA cap, we determined the effects of amino acid substitutions in eIF4E and VP39 cap-binding sites on their affinity for m(7)GDP. The requirements for residues suggested to pi-pi stack and hydrogen bond with the m(7)G base were examined in each protein by measuring their affinities for m(7)GDP by fluorimetry. The results suggest that both eIF4E and VP39 require a complicated pattern of both orientation and identity of the stacking aromatic residues to permit the selective binding of m(7)GDP.

Targeting of the zymogen-granule protein syncollin in AR42J and AtT-20 cells.

Syncollin is a 13-kDa protein associated with the membranes of pancreatic zymogen granules. Here we determine the in situ localization of syncollin in pancreatic acinar cells from adult and neonatal rats, and study the targeting of green fluorescent protein-(GFP-) and His(6)-tagged syncollin chimaeras in model exocrine and endocrine secretory cells. Immunocytochemical analysis of the distribution of syncollin in fully differentiated and neonatal acinar cells revealed a granular pattern that corresponded with that of the zymogen-granule markers synaptobrevin 2 and amylase. In fully differentiated acinar cells syncollin-positive vesicles were detected in the apical region of the cells, whereas in neonatal acinar cells they were found clustered near the cell nucleus. Both GFP- and His(6)-tagged syncollin entered the secretory pathway when transiently expressed in AR42J or AtT-20 cells. Syncollin-GFP was found predominantly in amylase-positive granules in AR42J cells and in adrenocorticotrophic hormone- (ACTH-) positive granules in AtT-20 cells. Syncollin-GFP was also present in the Golgi complex in AR42J cells. Syncollin-His(6) became localized in ACTH-containing granules in the neuritic processes of AtT-20 cells. In AR42J cells syncollin-His(6) did not co-localize with amylase, but was detected in acidic vesicles. These results show that the exocrine protein syncollin contains intrinsic cell-type-independent targeting information that is retained in both exocrine and endocrine cells after fusion to the GFP tag. In contrast, His(6)-tagged syncollin is efficiently targeted to secretory granules only in AtT-20 cells and not in AR42J cells.

Quantitative analysis of nuclear localization signal (NLS)-importin alpha interaction through fluorescence depolarization. Evidence for auto-inhibitory regulation of NLS binding.

We have developed a quantitative in vitro steady-state fluorescence depolarization assay to measure the interaction of a nuclear localization signal (NLS) substrate with its receptors. This assay relies on the change in fluorescence depolarization of an NLS fused to the green fluorescent protein upon binding to receptor. No binding is observed in the absence of a functional NLS, and binding affinities measured correlate with previous in vivo studies of NLS function. We have used this assay to test an auto-inhibitory model for the interaction of an NLS with the NLS receptor complex. This model suggests that NLS binding to importin alpha is modulated by an auto-inhibitory sequence within the N terminus of importin alpha, which is displaced by importin beta binding. Consistent with this model, NLS substrates bind tightly to an N-terminally truncated importin alpha lacking the auto-inhibitory domain (K(d) approximately 10 nm), but measurable binding to full-length importin alpha is only observed upon addition of importin beta. Our quantitative results support the auto-inhibitory model and suggest a mechanism for a switch between a cytoplasmic, high affinity and a nuclear, low affinity NLS receptor. This predicted mode of interaction would facilitate binding of substrate in the cytoplasm and its subsequent release into the nucleus.

Analysis of the association of syncollin with the membrane of the pancreatic zymogen granule.

Syncollin is a pancreatic zymogen granule protein that was isolated through its ability to bind to syntaxin. Here we show that syncollin has a cleavable signal sequence and can be removed from granule membranes by washing with sodium carbonate. When membranes were subjected to Triton X-114 partitioning, syncollin was found predominantly in the aqueous phase, indicating that it is not sufficiently hydrophobic to be embedded in the membrane. Syncollin has intramolecular disulfide bonds and was accessible to water-soluble cross-linking and biotinylating reagents only when granules were lysed by sonication. These results indicate that syncollin is tightly bound to the luminal surface of the granule membrane. In situ, syncollin was resistant to proteases such as trypsin. When granule membranes were solubilized in ionic detergents such as deoxycholate, this trypsin resistance was maintained, and syncollin migrated on sucrose density gradients as a large (150 kDa) protein. In contrast, in non-ionic detergents such as Triton X-100, syncollin became partially sensitive to trypsin and behaved as a monomer. Syncollin in alkaline extracts of granule membranes was also monomeric. However, reduction of the pH regenerated the oligomeric form, which was insoluble. We conclude that syncollin exists as a homo-oligomer and that its ability to self-associate can be reversibly modulated via changes in pH. In light of our findings, we reassess the likely role of syncollin in the pancreatic acinar cell.

mRNA cap recognition: dominant role of enhanced stacking interactions between methylated bases and protein aromatic side chains.

We have determined, by high resolution x-ray analysis, 10 structures comprising the mRNA cap-specific methyltransferase VP39 or specific mutants thereof in the presence of methylated nucleobase analogs (N1-methyladenine, N3-methyladenine, N1-methylcytosine, N3-methylcytosine) and their unmethylated counterparts, or nucleoside N7-methylguanosine. Together with solution affinity studies and previous crystallographic data for N7-methylguanosine and its phosphorylated derivatives, these data demonstrate that only methylated, positively charged bases are bound, indicating that their enhanced stacking with two aromatic side chains of VP39 (Tyr 22 and Phe 180) plays a dominant role in cap recognition. Four key features characterize this stacking interaction: (i) near perfect parallel alignment between the sandwiched methylated bases and aromatic side chains, (ii) substantial areas of overlap in the two-stacked rings, (iii) a 3.4-A interplanar spacing within the overlapping region, and (iv) positive charge in the heterocyclic nucleobase.

SNAP-25.

SNAP-25 belongs to a family of evolutionarily conserved proteins whose members are essential for exocytosis. Neurons and neuroendocrine cells differentially express two SNAP-25 isoforms in a developmentally regulated manner, and related homologues have been detected in most eukaryotic cells. SNAP-25 is localised on the cytoplasmic face of the plasma membrane and on secretory vesicles. It forms a stable ternary complex with two other exocytotic proteins: syntaxin and the synaptic vesicle protein synaptobrevin. A cytosolic ATPase dissociates this complex during priming of the exocytotic apparatus. Subsequent reassembly is promoted by SNAP-25 and may drive Ca(2+)-triggered vesicle-plasma membrane fusion. A mutant mouse that lacks the SNAP-25 gene is defective in neuronal dopamine signalling and exhibits similar behaviour as sufferers from hyperactivity disorders. Use of this animal model thus provides a promising avenue for the development of therapeutic treatments. Additionally, SNAP-25-based peptides that mimic the effect of botulinum neurotoxin A may be used for the treatment of involuntary muscle spasms.

Structural basis for sequence-nonspecific recognition of 5'-capped mRNA by a cap-modifying enzyme.

Sequence-nonspecific binding of RNA, recognition of a 7-methylguanosine 5' mRNA cap, and methylation of a nucleic acid backbone are three crucial and ubiquitous events in eukaryotic nucleic acid processing and function. These three events occur concurrently in the modification of vaccinia transcripts by the methyltransferase VP39. We report the crystal structure of a ternary complex comprising VP39, coenzyme product S-adenosylhomocysteine, and a 5' m7 G-capped, single-stranded RNA hexamer. This structure reveals a novel and general mechanism for sequence-non-specific recognition of the mRNA transcript in which the protein interacts solely with the sugar-phosphate backbone of a short, single-stranded RNA helix. This report represents the first direct and detailed view of a protein complexed with single-stranded RNA or 5'-capped mRNA.

Evidence that the RNA methylation and poly(A) polymerase stimulatory activities of vaccinia virus protein VP39 do not impinge upon one another.

Vaccinia protein VP39 has two RNA modifying activities. In monomeric form, it acts as an mRNA cap-specific 2'-O-methyltransferase, specifically modifying the ribose moiety of the first transcribed nucleotide of m7G-capped mRNA. In association with VP55, the catalytic subunit of the vaccinia poly(A) polymerase, VP39 facilitates the rapid elongation of poly(A) tails that are already greater than approximately 35 nt in length. Introducing new assays, we provide evidence that substrates for each of VP39's two activities do not detectably modulate the converse reaction and that VP39's 2'-O-methyltransferase activity is not significantly affected by its association with VP55. In an electrophoretic mobility shift assay, VP39 interacted with a short (5 nucleotide) RNA only when the latter was m7G-capped. Complexes with longer (22 nucleotide) RNAs were more stable (i.e., cap-independent) but were further stabilized by the presence of an m7G cap. An additional complex was observed at elevated RNA:protein molar ratios, indicating the presence of two RNA binding sites per VP39 molecule. Interaction at one of these sites was stabilized by the cap structure. Additional experiments indicated that RNA molecules undergoing poly(A) tail elongation by the VP55-VP39 heterodimer are not favored as cap-methylation substrates.

Recognition of capped RNA substrates by VP39, the vaccinia virus-encoded mRNA cap-specific 2'-O-methyltransferase.

We have investigated the interaction of VP39, the vaccinia-encoded mRNA cap-specific 2'-O-methyltransferase, with its capped RNA substrate. Two sites on the protein surface, responsible for binding the terminal cap nucleotide (m7G) and cap-proximal RNA, were characterized, and a third (downstream RNA binding) site was identified. Regarding the crystallographically defined m7G binding pocket, VP39 showed significant activity with adenine-capped RNA. Although VP39 mutants lacking specific m7G-contact side chains within the pocket showed reduced catalytic activity, none was transformed into a cap-independent RNA methyltransferase. Moreover, each retained a preference for m7G and A over unmethylated G as the terminal cap nucleotide, indicating a redundancy of m7G-contact residues able to confer cap-type specificity. Despite containing the 2'-O-methylation site, m7GpppG (cap dinucleotide) could not be methylated by VP39, but m7GpppGUbiotinp could. This indicated the minimum-length 2'-O-methyltransferase substrate to be either m7GpppGp, m7GpppGpN, or m7GpppGpNp. RNA-protein contacts immediately downstream of the m7GpppG moiety were found to be pH-sensitive. This was detectable only in the context of a weakened interaction of near-minimum-length substrates with VP39's m7G binding pocket (through the use of either adenine-capped substrate or a VP39 pocket mutant), as a dramatic elevation of KM at pH values above 7.5. KM values for substrates with RNA chain lengths of 2-6 nt were between 160 and 230 nM, but dropped to 9-15 nM upon increasing chain lengths to 20-50 nt. This suggested the binding of regions of the RNA substrate >6 nt from the 5' terminus to a previously unknown site on the VP39 surface.

Disruption of syntaxin-mediated protein interactions blocks neurotransmitter secretion.

The membrane protein syntaxin participates in several protein-protein interactions that have been implicated in neurotransmitter release. To probe the physiological importance of these interactions, we microinjected into the squid giant presynaptic terminal botulinum toxin C1, which cleaves syntaxin, and the H3 domain of syntaxin, which mediates binding to other proteins. Both reagents inhibited synaptic transmission yet did not affect the number or distribution of synaptic vesicles at the presynaptic active zone. Recombinant H3 domain inhibited the interactions between syntaxin and SNAP-25 that underlie the formation of stable SNARE complexes in vitro. These data support the notion that syntaxin-mediated SNARE complexes are necessary for docked synaptic vesicles to fuse.

In vitro exocytosis in sea urchin eggs requires a synaptobrevin-related protein.

Sea urchin eggs provide an efficient in vitro model of exocytosis. We have identified proteins in sea urchin eggs that cross-react with antibodies to mammalian synaptobrevin, synaptotagmin, SNAP-25, syntaxin and rab3a. We show that these proteins are localized to the sea urchin egg cortex, using western blotting and immunocytochemistry. Tetanus toxin light chain cleaves the synaptobrevin-related protein in vitro and inhibits calcium-induced exocytosis. These data demonstrate a conservation between phyla of protein sequence and molecular mechanisms thought to facilitate exocytosis and show that the sea urchin egg provides a unique in vitro exocytotic model with which to study the conserved protein machinery of membrane fusion during secretion.

Specific protein recognition of an mRNA cap through its alkylated base.

The specific binding of N7-methylguanine cap analogues to the RNA methyltransferase VP39 was observed through X-ray crystallography, providing a prototypical structure for a complex between a protein and an mRNA 5' cap.

Comparison of parametrized models for computer-based estimation of diabetic patient glucose response.

Two mathematical models for the description of diabetic patient glucose behaviour are proposed. Unlike high order differential-equation based compartmental models, these models employ only the data typically available to a diabetic patient: the history of measured blood glucose concentrations and of insulin injections. The model structures are compared with a native benchmark (zero-order hold) model in a computer simulation. It is demonstrated that, given four daily blood glucose measurements and two daily insulin injections, a parametrized model of patient blood glucose response to insulin can provide relevant data in the estimation of a patient's future blood glucose response in terms of past blood glucose measurements and insulin injections. Parametrized model root means squared errors of glycaemic predictions for 18 simulated patients ranged from 7-22 mg dl-1, as compared with 19-42 mg dl-1 for the benchmark model.