Antigen presentation subverted: Structure of the human cytomegalovirus protein US2 bound to the class I molecule HLA-A2

Many persistent viruses have evolved the ability to subvert MHC class I antigen presentation. Indeed, human cytomegalovirus (HCMV) encodes at least four proteins that down-regulate cell-surface expression of class I. The HCMV unique short (US)2 glycoprotein binds newly synthesized class I molecules within the endoplasmic reticulum (ER) and subsequently targets them for proteasomal degradation. We report the crystal structure of US2 bound to the HLA-A2/Tax peptide complex. US2 associates with HLA-A2 at the junction of the peptide-binding region and the α3 domain, a novel binding surface on class I that allows US2 to bind independently of peptide sequence. Mutation of class I heavy chains confirms the importance of this binding site in vivo. Available data on class I-ER chaperone interactions indicate that chaperones would not impede US2 binding. Unexpectedly, the US2 ER-luminal domain forms an Ig-like fold. A US2 structure-based sequence alignment reveals that seven HCMV proteins, at least three of which function in immune evasion, share the same fold as US2. The structure allows design of further experiments to determine how US2 targets class I molecules for degradation.

Dynamics of Immature Secretory Granules: Role of Cytoskeletal Elements during Transport, Cortical Restriction, and F-Actin-dependent Tethering

Secretory granules store neuropeptides and hormones and exhibit regulated exocytosis upon appropriate cellular stimulation. They are generated in the trans-Golgi network as immature secretory granules, short-lived vesicular intermediates, which undergo a complex and poorly understood maturation process. Due to their short half-life and low abundance, real-time studies of immature secretory granules have not been previously possible. We describe here a pulse/chase-like system based on the expression of a human chromogranin B-GFP fusion protein in neuroendocrine PC12 cells, which permits direct visualization of the budding of immature secretory granules and their dynamics during maturation. Live cell imaging revealed that newly formed immature secretory granules are transported in a direct and microtubule-dependent manner within a few seconds to the cell periphery. Our data suggest that the cooperative action of microtubules and actin filaments restricts immature secretory granules to the F-actin-rich cell cortex, where they move randomly and mature completely within a few hours. During this maturation period, secretory granules segregate into pools of different motility. In a late phase of maturation, 60% of secretory granules were found to be immobile and about half of these underwent F-actin-dependent tethering.

Activation of the Maize Anthocyanin Gene a2 Is Mediated by an Element Conserved in Many Anthocyanin Promoters1

Two transcription factors, C1 (a Myb-domain protein) and B (a basic-helix-loop-helix protein), mediate transcriptional activation of the anthocyanin-biosynthetic genes of maize (Zea mays). To begin to assess the mechanism of activation, the sequences required for C1- and B-mediated induction have been determined for the a2 promoter, which encodes an anthocyanin-biosynthetic enzyme. Analysis of a series of 7- to 13-base-pair substitutions revealed two regions crucial for activation. One region, centered at −99, contained a C1-binding site that abolished C1 binding. The other crucial region was adjacent, centered at −91. C1 binding was not detected at this site, and mutation of this site did not prevent C1 binding at −99. An oligonucleotide dimer containing these two crucial elements was sufficient for C1 and B activation of a heterologous promoter. These data suggest that activation of the anthocyanin genes involves C1 and another factor binding at closely adjacent sites. Mutating a previously postulated anthocyanin consensus sequence within a2 did not significantly reduce activation by C1 and B. However, sequence comparisons of the crucial a2 regions with sequences important for C1- and B-mediated activation in two other anthocyanin promoters led to a revised consensus element shared by these promoters.

Molecular and Enzymatic Characterization of Three Phosphoinositide-Specific Phospholipase C Isoforms from Potato1

Many cellular responses to stimulation of cell-surface receptors by extracellular signals are transmitted across the plasma membrane by hydrolysis of phosphatidylinositol-4,5-bisphosphate (PIP2), which is cleaved into diacylglycerol and inositol-1,4,5-tris-phosphate by phosphoinositide-specific phospholipase C (PI-PLC). We present structural, biochemical, and RNA expression data for three distinct PI-PLC isoforms, StPLC1, StPLC2, and StPLC3, which were cloned from a guard cell-enriched tissue preparation of potato (Solanum tuberosum) leaves. All three enzymes contain the catalytic X and Y domains, as well as C2-like domains also present in all PI-PLCs. Analysis of the reaction products obtained from PIP2 hydrolysis unequivocally identified these enzymes as genuine PI-PLC isoforms. Recombinant StPLCs showed an optimal PIP2-hydrolyzing activity at 10 μm Ca2+ and were inhibited by Al3+ in equimolar amounts. In contrast to PI-PLC activity in plant plasma membranes, however, recombinant enzymes could not be activated by Mg2+. All three stplc genes are expressed in various tissues of potato, including leaves, flowers, tubers, and roots, and are affected by drought stress in a gene-specific manner.

Homotypic Fusion of Immature Secretory Granules During Maturation Requires Syntaxin 6

Homotypic fusion of immature secretory granules (ISGs) gives rise to mature secretory granules (MSGs), the storage compartment in endocrine and neuroendocrine cells for hormones and neuropeptides. With the use of a cell-free fusion assay, we investigated which soluble N-ethylmaleimide-sensitive fusion protein attachment receptor (SNARE) molecules are involved in the homotypic fusion of ISGs. Interestingly, the SNARE molecules mediating the exocytosis of MSGs in neuroendocrine cells, syntaxin 1, SNAP-25, and VAMP2, were not involved in homotypic ISG fusion. Instead, we have identified syntaxin 6 as a component of the core machinery responsible for homotypic ISG fusion. Subcellular fractionation studies and indirect immunofluorescence microscopy show that syntaxin 6 is sorted away during the maturation of ISGs to MSGs. Although, syntaxin 6 on ISG membranes is associated with SNAP-25 and SNAP-29/GS32, we could not find evidence that these target (t)-SNARE molecules are involved in homotypic ISG fusion. Nor could we find any involvement for the vesicle (v)-SNARE VAMP4, which is known to be associated with syntaxin 6. Importantly, we have shown that homotypic fusion requires the function of syntaxin 6 on both donor as well as acceptor membranes, which suggests that t–t-SNARE interactions, either direct or indirect, may be required during fusion of ISG membranes.

Secretory granule targeting of atrial natriuretic peptide correlates with its calcium-mediated aggregation.

Atrial natriuretic peptide (ANP) is a 28-aa peptide hormone secreted predominantly from atrial cardiocytes. ANP is first synthesized in the form of a 126-aa precursor (proANP) which is targeted to dense core granules of the regulated secretory pathway. ProANP is stored until the cell receives a signal that triggers the processing and release of the mature peptide (regulated secretion). Various models have been proposed to explain the targeting of selected proteins to the regulated secretory pathway, including specific "sorting receptors" and calcium-mediated aggregation. As potential calcium binding regions had previously been reported in the profragment of ANP, the current study was undertaken in an effort to determine the relationship between the ability of ANP to enter the regulated secretory pathway and its calcium-mediated aggregation. Deletion and site-directed mutagenesis of selected regions of the prosegment demonstrates that acidic amino acids at positions 23 and 24 are critical for both regulated secretion of proANP from transfected AtT-20 cells and calcium-mediated aggregation of purified recombinant proANP in vitro. These results demonstrate that the ability of certain proteins to enter secretory granules is directly linked to their calcium-mediated aggregation.

Perturbations in maturation of secretory proteins and their association with endoplasmic reticulum chaperones in a cell culture model for epithelial ischemia.

The effects of ischemia on the maturation of secretory proteins are not well understood. Among several events that occur during ischemia-reperfusion are a rapid and extensive decrease in ATP levels and an alteration of cellular oxidative state. Since the normal folding and assembly of secretory proteins are mediated by endoplasmic reticulum (ER) molecular chaperones, the function of which depends on ATP and maintenance of an appropriate redox environment, ischemia might be expected to perturb folding of secretory proteins. In this study, whole animal and cultured cell models for the epithelial ischemic state were used to examine this possibility. After acute kidney ischemia, marked increases in the mRNA levels of the ER chaperones glucose-regulated protein (grp)78/immunoglobulin-binding protein (BiP), grp94, and ER protein (ERp)72 were noted. Likewise, when cellular ATP was depleted to less than 10% of control with antimycin A, mRNA levels of BiP, ERp72, and grp94 were increased in kidney and thyroid epithelial cell culture models. Since the signal for the up-regulation of these stress proteins is believed to be the accumulation of misfolded/misassembled secretory proteins in the ER, their induction after ischemia in vivo and antimycin treatment of cultured cells suggests that maturation of secretory proteins in the ER lumen might indeed be perturbed. To analyze the effects of antimycin A on the maturation of secretory proteins, we studied the fate of thyroglobulin (Tg), a large oligomeric secretory glycoprotein, the folding and assembly of which seems to require a variety of ER chaperones. Treatment of cultured thyroid epithelial cells with antimycin A greatly inhibited ( > 90%) the secretion of Tg. Sucrose density gradient analysis revealed that in antimycin A-treated cells Tg associates into large macromolecular complexes which, by immunofluorescence, appeared to localize to the ER. Furthermore, coimmunoprecipitation studies after antimycin A treatment demonstrated that Tg stably associates with BiP, grp94, and ERp72. Together, our results suggest that a key cellular lesion in ischemia is the misfolding of secretory proteins as they transit the ER, and this leads not only to increased expression of ER chaperones but also to their stable association with and the subsequent retention of at least some misfolded secretory proteins.

Haemonchus contortus GA1 antigens: related, phospholipase C-sensitive, apical gut membrane proteins encoded as a polyprotein and released from the nematode during infection.

It was previously shown that the Haemonchus contortus apical gut surface proteins p46, p52, and p100 induced protective immunity to challenge infections in goats. Here, it is shown that the three proteins are all encoded by a single gene (GA1) and initially expressed in adult parasites as a polyprotein (p100GA1). p46GA1 and p52GA1 are related proteins with 47% sequence identity, including a cysteine-containing region, which appears to confer secondary structure to these proteins, and a region with sequence similarity to bacterial Tolb proteins. GA1 protein expression is regulated during the life cycle at the level of transcript abundance. Only p52GA1 has characteristics of a glycosylinositolphospholipid membrane-anchored protein. However, both p46GA1 and p52GA1 were released from the gut membrane by phosphatidylinositol specific-phospholipase C, suggesting that p46GA1 membrane association depends on interactions with a glycosylinositolphospholipid gut membrane protein. Finally, GA1 proteins occur in abomasal mucus of infected lambs, demonstrating possible presentation to the host immune system during H. contortus infection. The results identify multiple characteristics of the GA1 proteins that should be considered for design of recombinant antigens for vaccine trials and that implicate a series of cellular processes leading to modification and expression of GA1 proteins at the nematode apical gut surface.

Enhanced phospholipase C-gamma1 activity produced by association of independently expressed X and Y domain polypeptides.

The X and Y domains of phospholipase C (PLC)-gamma1, which are conserved in all mammalian phosphoinositide-specific PLC isoforms and are proposed to interact to form the catalytic site, have been expressed as individual hexahistidine-tagged fusion proteins in the baculovirus system. Following coinfection of insect cells with recombinant viruses, association of X and Y polypeptides was demonstrated in coprecipitation assays. When enzyme activity was examined, neither domain possessed catalytic activity when expressed alone; however, coexpression of the X and Y polypeptides produced a functional enzyme. This reconstituted phospholipase activity remained completely dependent on the presence of free Ca2+. The specific activity of the X:Y complex was significantly greater (20- to 100-fold) than that of holoPLC-gamma1 and was only moderately influenced by varying the concentration of substrate. The enzyme activities of holoPLC-gamma1 and the X:Y complex exhibited distinct pH optima. For holoPLC-gamma1 maximal activity was detected at pH 5.0, while activity of the X:Y complex was maximal at pH 7.2.

The yeast and mammalian isoforms of phosphatidylinositol transfer protein can all restore phospholipase C-mediated inositol lipid signaling in cytosol-depleted RBL-2H3 and HL-60 cells.

The mammalian phosphatidylinositol transfer proteins (PITP) and the yeast Saccharomyces cerevisiae PITP (SEC14p) that show no sequence homology both catalyze exchange of phosphatidylinositol (PI) between membranes compartments in vitro. In HL-60 cells where the cytosolic proteins are depleted by permeabilization, exogenously added PITPalpha is required to restore G protein-mediated phospholipase Cbeta (PLCbeta) signaling. Recently, a second mammalian PITPbeta form has been described that shows 77% identity to rat PITPalpha. We have examined the ability of the two mammalian PITPs and SEC14p to restore PLC-mediated signaling in cytosol-depleted HL-60 and RBL-2H3 cells. Both PITPalpha and PITPbeta isoforms as well as SEC14p restore G protein-mediated PLCbeta signaling with a similar potency. In RBL-2H3 cells, crosslinking of the IgE receptor by antigen stimulates inositol lipid hydrolysis by tyrosine phosphorylation of PLCgamma1. Permeabilization of RBL cells leads to loss of PLCgamma1 as well as PITP into the extracellular medium and this coincides with loss of antigen-stimulated lipid hydrolysis. Both PLCgamma1 and PITP were required to restore inositol lipid signaling. We conclude that (i) because the PI binding/transfer activities of PITP/SEC14p is the common feature shared by all three transfer proteins, it must be the relevant activity that determines their abilities to restore inositol lipid-mediated signaling and (ii) PITP is a general requirement for inositol lipid hydrolysis regardless of how and which isoform of PLC is activated by the appropriate agonist.

Different functions for the interleukin 8 receptors (IL-8R) of human neutrophil leukocytes: NADPH oxidase and phospholipase D are activated through IL-8R1 but not IL-8R2.

Two monoclonal antibodies, anti-IL8R1 and anti-IL8R2, raised against both interleukin 8 receptors (IL-8R) of human neutrophils, IL-8R1 and IL-8R2, were used to study individual receptor functions after stimulation with IL-8, GRO alpha, or NAP-2. Efficacy and selectivity of the antibodies were tested in Jurkat cells transfected with cDNA coding for one or the other receptor. The binding of 125 I labeled IL-8 and IL-8-induced changes of the cytosolic free Ca2+ concentration were inhibited by anti-IL8RI in cells expressing IL-8R1 and by anti-IL8R2 in cells expressing IL-8R2. In human neutrophils, release of elastase was observed after stimulation with IL-8 or GRO alpha. The response to IL-8 was inhibited slightly by anti-IL8R1 and more substantially when both monoclonal antibodies were present, while the response to GRO alpha was inhibited by anti-IL8R2 but was not affected by anti-IL8R1. These results indicate that both IL-8 receptors can signal independently for granule enzyme release. Superoxide production, a measure of the respiratory burst, was obtained with increasing concentrations of IL-8 with maximum effects at 25 to 50 nM, but no response was observed upon challenge with GRO alpha or NAP-2 up to 1000 nM. The superoxide production induced by IL-8 was inhibited by anti-IL8R1, but was not affected by anti-IL8R2. Stimulation of neutrophils with IL-8, in contrast to GRO alpha or NAP-2, also elicited phospholipase D activity. The effect of IL-8 was again inhibited by anti-IL-8R1 but not by anti-IL8R2, indicating that this response, like the respiratory burst, was mediated by IL-8R1. Taken together, our results show that IL-8R1 and IL-8R2 are functionally different. Responses, such as cytosolic free Ca2+ changes and the release of granule enzymes, are mediated through both receptors, whereas the respiratory burst and the activation of phospholipase D depend exclusively on stimulation through IL-8R1.

Antigenic peptides containing large PEG loops designed to extend out of the HLA-A2 binding site form stable complexes with class I major histocompatibility complex molecules.

Recognition of peptides bound to class I major histocompatibility complex (MHC) molecules by specific receptors on T cells regulates the development and activity of the cellular immune system. We have designed and synthesized de novo cyclic peptides that incorporate PEG in the ring structure for binding to class I MHC molecules. The large PEG loops are positioned to extend out of the peptide binding site, thus creating steric effects aimed at preventing the recognition of class I MHC complexes by T-cell receptors. Peptides were synthesized and cyclized on polymer support using high molecular weight symmetrical PEG dicarboxylic acids to link the side chains of lysine residues substituted at positions 4 and 8 in the sequence of the HLA-A2-restricted human T-lymphotrophic virus type I Tax peptide. Cyclic peptides promoted the in vitro folding and assembly of HLA-A2 complexes. Thermal denaturation studies using circular dichroism spectroscopy showed that these complexes are as stable as complexes formed with antigenic peptides.

Mammalian phospholipase D: phosphatidylethanolamine as an essential component.

Bovine kidney phospholipase D (PLD) was assayed by measuring the formation of phosphatidylethanol from added radioactive phosphatidylcholine (PtdCho) in the presence of ethanol, guanosine 5'-[gamma-thio]triphosphate, ammonium sulfate, and cytosol factor that contained small GTP-binding regulatory proteins. The PLD enzyme associated with particulate fractions was solubilized by deoxycholate and partially purified by chromatography on a heparin-Sepharose column. This PLD preferentially used PtdCho as substrate. After purification, the enzyme per se showed little or practically no activity but required an additional factor for the enzymatic reaction. This factor was extracted with chloroform/methanol directly from particulate fractions of various tissues, including kidney, liver, and brain, and identified as phosphatidylethanolamine (PtdEtn), although this phospholipid did not serve as a good substrate. Plasmalogen-rich PtdEtn, dioleoyl-PtdEtn, and L-alpha-palmitoyl-beta-linoleoyl-PtdEtn were effective, but dipalmitoyl-PtdEtn was inert. Sphingomyelin was 30% as active as PtdEtn. The results suggest that mammalian PLD reacts nearly selectively with PtdCho in the form of mixed micelles or membranes with other phospholipids, especially PtdEtn.

Chromogranin B (secretogranin I) promotes sorting to the regulated secretory pathway of processing intermediates derived from a peptide hormone precursor.

Chromogranin B (CgB, secretogranin I) is a widespread constituent of neuroendocrine secretory granules whose function is unknown. To determine whether CgB affects the sorting of peptide hormone and neuropeptide precursors to secretory granules, we overexpressed CgB in AtT-20 cells, which exhibit an only moderate capacity to sort proopiomelanocortin and proteolytic fragments derived therefrom. In mock-transfected AtT-20 cells, a substantial proportion of newly synthesized proopiomelanocortin and its two primary proteolytic products generated in the trans-Golgi network, the N-terminal 23-kDa fragment containing adrenocorticotropin and the C-terminal beta-lipotropin fragment, was secreted via the constitutive pathway. Two- to three-fold overexpression of CgB markedly reduced the constitutive secretion of the 23-kDa fragment, but not beta-lipotropin and tripled the amount of adrenocorticotropin generated and stored in secretory granules. Our results indicate the existence of neuroendocrine-specific helper proteins which promote the sorting from the trans-Golgi network to secretory granules of certain processing intermediates derived from peptide hormone and neuropeptide precursors and demonstrate that CgB functions as such.

Inositol 1,4,5-trisphosphate receptor subtype 3 in pancreatic islet cell secretory granules revisited.

It has been reported that the inositol 1,4,5-trisphosphate receptor subtype 3 is expressed in islet cells and is localized to both insulin and somatostatin granules [Blondel, O., Moody, M. M., Depaoli, A. M., Sharp, A. H., Ross, C. A., Swift, H. & Bell, G. I. (1994) Proc. Natl. Acad. Sci. USA 91, 7777-7781]. This subcellular localization was based on electron microscope immunocytochemistry using antibodies (affinity-purified polyclonal antiserum AB3) directed to a 15-residue peptide of rat inositol trisphosphate receptor subtype 3. We now show that these antibodies cross-react with rat, but not human, insulin. Accordingly, the anti-inositol trisphosphate receptor subtype 3 (AB3) antibodies label electron dense cores of mature (insulin-rich) granules of rat pancreatic beta cells, and rat granule labeling was blocked by preabsorption of the AB3 antibodies with rat insulin. The immunostaining of immature, Golgi-associated proinsulin-rich granules with AB3 antibodies was very weak, indicating that cross-reactivity is limited to the hormone and not its precursor. Also, the AB3 antibodies labeled pure rat insulin crystals grown in vitro but failed to stain crystals grown from pure human insulin. By immunoprecipitation, the antibodies similarly displayed a higher affinity for rat than for human insulin. We could not confirm the labeling of somatostatin granules using AB3 antibodies.