HA Epitope Research Articles

Intracellular delivery of peptides via association with ubiquitin or SUMO-1 coupled to protein transduction domains

BackgroundWe previously developed small hybrid proteins consisting of SUMO-1 linked to an heptapeptide fused to the Tat protein transduction domain (PTD). The heptapeptide motif was selected from a library of random sequences to specifically bind HIV-1 regulatory proteins Tat or Rev. These constructs, named SHP, are able to enter primary lymphocytes and some of them inhibit HIV-1 replication. Considering these positive results and other data from the literature, we further tested the ability of ubiquitin or SUMO-1 linked to various PTD at their N-terminus to deliver within cells proteins or peptides fused downstream of their diglycine motif. In this system it is expected that the intracellular ubiquitin or SUMO-1 hydrolases cleave the PTD-Ub or PTD-SUMO-1 modules from the cargo polypeptide, thereby allowing its delivery under an unmodified form.ResultsSeveral bacterial expression vectors have been constructed to produce modular proteins containing from the N- to the C-terminus: the FLAG epitope, a cleavage site for a protease, a PTD, human ubiquitin or SUMO-1, and either GFP or the HA epitope. Nine different PTDs were tested, including the Tat basic domain, wild type or with various mutations, and stretches of arginine or lysine. It was observed that some of these PTDs, mainly the Tat PTD and seven or nine residues long polyarginine motifs, caused association of the hybrid proteins with cells, but none of these constructs were delivered to the cytosol. This conclusion was derived from biochemical and immunofluorescence studies, and also from the fact that free cargo protein resulting from cleavage by proteases after ubiquitin or SUMO-1 was never observed. However, in agreement with our previous observations, mutation of the diglycine motif into alanine-arginine, as in the SHP constructs, allows cytosol entry demonstrated by immunofluorescence observations on living cells and by cell fractionation analyses. This process results from a non-endocytic pathway.ConclusionOur observations indicate that fusion of SUMO-1 to a peptide-PTD module allows generation of a stable hybrid protein that is easily produced in bacteria and which efficiently enters into cells but this property necessitates mutation of the diglycine motif at the end of SUMO-1, thereby impairing delivery of the peptide alone.

Mind Bomb-2 Is an E3 Ligase That Ubiquitinates the N-Methyl-d-aspartate Receptor NR2B Subunit in a Phosphorylation-dependent Manner

The N-methyl-D-aspartate receptor (NMDAR) plays a critical role in synaptic plasticity. Post-translational modifications of NMDARs, such as phosphorylation, alter both the activity and trafficking properties of NMDARs. Ubiquitination is increasingly being recognized as another post-translational modification that can alter synaptic protein composition and function. We identified Mind bomb-2 as an E3 ubiquitin ligase that interacts with and ubiquitinates the NR2B subunit of the NMDAR in mammalian cells. The protein-protein interaction and the ubiquitination of the NR2B subunit were found to be enhanced in a Fyn phosphorylation-dependent manner. Immunocytochemical studies reveal that Mind bomb-2 is localized to postsynaptic sites and colocalizes with the NMDAR in apical dendrites of hippocampal neurons. Furthermore, we show that NMDAR activity is down-regulated by Mind bomb-2. These results identify a specific E3 ubiquitin ligase as a novel interactant with the NR2B subunit and suggest a possible mechanism for the regulation of NMDAR function involving both phosphorylation and ubiquitination.

The Activity of p53 Is Differentially Regulated by Brm- and Brg1-containing SWI/SNF Chromatin Remodeling Complexes

Brahma (Brm) and Brahma-related gene-1 (Brg1) ATPases share similarities in structure and function, but their presence in human SWI/SNF chromatin remodeling complexes is mutually exclusive. Although Brm and Brg1 can compensate for each other, it is possible that Brm and Brg1 have their unique properties to differentially regulate gene expression in vivo. To explore this, we examined the requirement of Brm and Brg1 for p53-dependent transcription, especially p53-mediated induction of p21 and MDM2, using cell lines in which Brm or Brg1 could be inducibly knocked down. We found that Brg1, but not Brm, is required for p21 induction in MCF7 cells. However, in Brg1-deficient H1299 cells, Brm is also required for p21 induction. Likewise, Brm is necessary for induction of p21 in MCF7 cells in which Brg1 is stably knocked down. In contrast, Brg1 has little, if any, effect on p53-mediated induction of MDM2 in cells that have Brm and vice versa. In addition, we demonstrated that the impaired induction of p21 upon Brg1 knockdown is at least in part due to decreased p53 binding to the p21 promoter. Taken together, we provided evidence that Brg1 is preferentially recruited by p53 for inducing a subset of target genes through chromatin remodeling. Thus, we hypothesize that the potential tumor suppressor function for Brg1 is mediated in part through the p53 pathway.

HFE Modulates Transferrin Receptor 2 Levels in Hepatoma Cells via Interactions That Differ from Transferrin Receptor 1-HFE Interactions

Mutations in the transmembrane glycoproteins transferrin receptor 2 (TfR2) and HFE are associated with hereditary hemochromatosis. Interactions between HFE and transferrin receptor 1 (TfR1) have been mapped to the alpha1- and alpha2-helices in HFE and to the helical domain of TfR1. Recently, TfR2 was also reported to interact with HFE in transfected mammalian cells. To test whether similar HFE residues are important for both TfR1 and TfR2 binding, a mutant form of HFE (W81AHFE) that has an approximately 5,000-fold lower affinity for TfR1 than HFE was employed. As expected, W81AHFE does not interact with TfR1. However, we found that the same mutation in HFE does not affect the TfR2/HFE interaction. This finding indicates that the TfR2/HFE and TfR1/HFE interactions are distinct. We further observed that, unlike TfR1/HFE, Tf does not compete with HFE for binding to TfR2 and that binding is independent of pH (pH 6-7.5). TfR2-TfR1 and HFE-HLA-B7 chimeras were generated to map the domains of the TfR2/HFE interaction. TfR1 and HLA-B7 were chosen because of their similar overall structures with TfR2 and HFE, respectively. We mapped the interacting domains to the putative stalk and protease-like domains of TfR2 located between residues 104 and 250 and to the alpha3 domain of HFE, both of which differ from the TfR1/HFE interacting domains. Furthermore, we found that HFE increases TfR2 levels in hepatic cells independent of holo-Tf.

Phosphatidylinositol 4-Phosphate 5-Kinase Reduces Cell Surface Expression of the Epithelial Sodium Channel (ENaC) in Cultured Collecting Duct Cells

Ubiquitination of ENaC subunits has been shown to negatively regulate the cell surface expression of ENaC channels. We have previously demonstrated that epsin links ubiquitinated ENaC to clathrin adaptors for clathrin-mediated endocytosis. Epsin is thought to directly modify the curvature of membranes upon binding to phosphatidylinositol 4,5-bisphosphate (PIP2) where it recruits clathrin and stimulates lattice assembly. Murine phosphatidylinositol 4-phosphate 5-kinase alpha (PI5KIalpha) has been shown to enhance endocytosis in a PIP2-dependent manner. We tested the hypothesis that PI5KIalpha-mediated PIP2 production would negatively regulate ENaC current by enhancing epsin-mediated endocytosis of the channel. Expression of PI5KIalpha decreased ENaC currents in Xenopus oocytes by 80%, entirely because of a decrease in cell surface ENaC levels. Catalytically inactive mutants of PI5Kalpha had no effect on ENaC activity. Expression of the PIP2 binding region of epsin increased ENaC current in oocytes, an effect completely reversed by co-expression of PI5KIalpha. Overexpression of epsin reduced amiloride-sensitive current in CCD cells. Overexpression of PI5KIalpha enhanced membrane PIP2 levels and reduced apical surface expression of ENaC in CCD cells, down-regulating amiloride-sensitive current. Knockdown of PI5KIalpha with isoform-specific siRNA resulted in a 4-fold enhancement of ENaC activity. PI5KIalpha localized exclusively to the apical plasma membrane domain when overexpressed in mouse CCD cells, consistent for a role in regulating PIP2 production at the apical plasma membrane. We conclude that membrane turnover events regulating ENaC surface expression and activity in oocytes and CCD cells can be regulated by PI5KIalpha.

Dimerization of the thyrotropin-releasing hormone receptor potentiates hormone-dependent receptor phosphorylation

The G protein-coupled thyrotropin (TSH)-releasing hormone (TRH) receptor forms homodimers. Regulated receptor dimerization increases TRH-induced receptor endocytosis. These studies test whether dimerization increases receptor phosphorylation, which could potentiate internalization. Phosphorylation at residues 355-365, which is critical for internalization, was measured with a highly selective phospho-site-specific antibody. Two strategies were used to drive receptor dimerization. Dimerization of a TRH receptor-FK506-binding protein (FKBP) fusion protein was stimulated by a dimeric FKBP ligand. The chemical dimerizer caused a large increase in TRH-dependent phosphorylation within 1 min, whereas a monomeric FKBP ligand had no effect. The dimerizer did not alter phoshorylation of receptors lacking the FKBP domain. Dimerization of receptors containing an N-terminal HA epitope also was induced with anti-HA antibody. Anti-HA IgG strongly increased TRH-induced phosphorylation, whereas monomeric Fab fragments had no effect. Anti-HA antibody did not alter phosphorylation in receptors lacking an HA tag. Furthermore, two phosphorylation-defective TRH receptors functionally complemented one another and permitted phosphorylation. Receptors with a D71A mutation in the second transmembrane domain do not signal, whereas receptors with four Ala mutations in the 355-365 region signal normally but lack phosphorylation sites. When D71A- and 4Ala-TRH receptors were expressed alone, neither underwent TRH-dependent phosphorylation. When they were expressed together, D71A receptor was phosphorylated by G protein-coupled receptor kinases in response to TRH. These results suggest that the TRH receptor is phosphorylated preferentially when it is in dimers or when preexisting receptor dimers are driven into microaggregates. Increased receptor phosphorylation may amplify desensitization.

Ubiquitination of β-Arrestin Links Seven-transmembrane Receptor Endocytosis and ERK Activation

Beta-arrestin2 and its ubiquitination play crucial roles in both internalization and signaling of seven-transmembrane receptors (7TMRs). To understand the connection between ubiquitination and the endocytic and signaling functions of beta-arrestin, we generated a beta-arrestin2 mutant that is defective in ubiquitination (beta-arrestin2(0K)), by mutating all of the ubiquitin acceptor lysines to arginines and compared its properties with the wild type and a stably ubiquitinated beta-arrestin2-ubiquitin (Ub) chimera. In vitro translated beta-arrestin2 and beta-arrestin2(0K) displayed equivalent binding to recombinant beta(2)-adrenergic receptor (beta(2)AR) reconstituted in vesicles, whereas beta-arrestin2-Ub bound approximately 4-fold more. In cellular coimmunoprecipitation assays, beta-arrestin2(0K) bound nonreceptor partners, such as AP-2 and c-Raf and scaffolded phosphorylated ERK robustly but displayed weak binding to clathrin. Moreover, beta-arrestin2(0K) was recruited only transiently to activated receptors at the membrane, did not enhance receptor internalization, and decreased the amount of phosphorylated ERK assimilated into isolated beta(2)AR complexes. Although the wild type beta-arrestin2 formed ERK signaling complexes with the beta(2)AR at the membrane, a stably ubiquitinated beta-arrestin2-Ub chimera not only stabilized the ERK signalosomes but also led to their endosomal targeting. Interestingly, in cellular fractionation assays, the ubiquitination state of beta-arrestin2 favors its distribution in membrane fractions, suggesting that ubiquitination increases the propensity of beta-arrestin for membrane association. Our findings suggest that although beta-arrestin ubiquitination is dispensable for beta-arrestin cytosol to membrane translocation and its "constitutive" interactions with some cytosolic proteins, it nevertheless is a prerequisite both for the formation of tight complexes with 7TMRs in vivo and for membrane compartment interactions that are crucial for downstream endocytic and signaling processes.

The ClC-3 Chloride Transport Protein Traffics through the Plasma Membrane via Interaction of an N-terminal Dileucine Cluster with Clathrin

ClC-3 is a ubiquitously expressed chloride transport protein that is present in synaptic vesicles and endosome/lysosome compartments. It is largely intracellular but has been observed at the plasma membrane as well. The aim of this study was to identify the pathways and regulation of ClC-3 trafficking to intracellular sites. At the steady state, approximately 94% of transfected ClC-3 was localized intracellularly, and only 6% was at the plasma membrane. Pulse labeling with [(35)S]methionine and biotinylation demonstrated that about 25% of newly synthesized ClC-3 traffics through the plasma membrane. We used both immunofluorescence microscopy and biotinylation assays to assess the trafficking of ClC-3. Plasma membrane ClC-3 was rapidly endocytosed (t((1/2)) approximately 9 min); a portion entered a recycling pool that returned to the cell surface after internalization, and the remainder trafficked to more distal intracellular compartments. ClC-3 associated with clathrin at the plasma membrane. Coimmunoprecipitation and glutathione S-transferase pulldown assays demonstrated that the N terminus of ClC-3 binds to clathrin. Alanine replacement of a dileucine acidic cluster within the cytosolic N terminus (amino acids 13-19) resulted in a molecule that had decreased endocytosis and increased surface expression. This replacement also abolished interaction with clathrin as assessed both by coimmunoprecipitation and glutathione S-transferase pulldown assays. We conclude that ClC-3 is primarily an intracellular transport protein that is transiently inserted into the plasma membrane where it is rapidly endocytosed. Internalization of ClC-3 depends on the interaction between an N-terminal dileucine cluster and clathrin.

Coactivators PGC-1β and SRC-1 Interact Functionally to Promote the Agonist Activity of the Selective Estrogen Receptor Modulator Tamoxifen

PGC-1beta is a transcriptional coactivator that enhances strongly and in a hormone-dependent manner the activity of the estrogen receptor alpha (ERalpha) while having only weak effects on similar steroid hormone receptors, such as ERbeta or the glucocorticoid receptor. Notably, PGC-1beta enhances ERalpha transcriptional activity not only in response to agonist ligands, such as estradiol, but also to selective ER modulators, such as tamoxifen. Here, we dissect the molecular mechanisms underlying the ability of PGC-1beta to act selectively on ERalpha and to promote the agonist activity of tamoxifen. We show that receptor selectivity is achieved by PGC-1beta interactions with not just the ligand binding domain (LBD), which is highly conserved among nuclear receptors, but also the N-terminal domain and the hinge/AF-2a region of ERalpha, which are less well conserved. PGC-1beta interacts directly with the hinge/AF-2a and LBD regions but indirectly and via the coactivator SRC-1 with the N-terminal domain. The three ERalpha surfaces and SRC-1 collectively enable efficient coactivation by PGC-1beta. Similar ERalpha surfaces and interactions enable PGC-1beta to coactivate transcription by tamoxifen-bound ERalpha. Surprisingly, PGC-1beta coactivation of tamoxifen-bound ERalpha depends partially on one of the LXXLL motifs of PGC-1beta and on Lys(362) of the ERalpha LBD (i.e. surfaces implicated in agonist-dependent interactions). Our findings suggest that tamoxifen-induced changes in the ERalpha LBD promote interactions with the coactivator PGC-1beta, which then cooperates with SRC-1 to enable tamoxifen agonism.

RAVE Is Essential for the Efficient Assembly of the C Subunit with the Vacuolar H+-ATPase

The RAVE complex is required for stable assembly of the yeast vacuolar proton-translocating ATPase (V-ATPase) during both biosynthesis of the enzyme and regulated reassembly of disassembled V(1) and V(0) sectors. It is not yet known how RAVE effects V-ATPase assembly. Previous work has shown that V(1) peripheral or stator stalk subunits E and G are critical for binding of RAVE to cytosolic V(1) complexes, suggesting that RAVE may play a role in docking of the V(1) peripheral stalk to the V(0) complex at the membrane. Here we provide evidence for an interaction between the RAVE complex and V(1) subunit C, another subunit that has been assigned to the peripheral stalk. The C subunit is unique in that it is released from both V(1) and V(0) sectors during disassembly, suggesting that subunit C may control the regulated assembly of the V-ATPase. Mutants lacking subunit C have assembly phenotypes resembling that of RAVE mutants. Both are able to assemble V(1)/V(0) complexes in vivo, but these complexes are highly unstable in vitro, and V-ATPase activity is extremely low. We show that in the absence of the RAVE complex, subunit C is not able to stably assemble with the vacuolar ATPase. Our data support a model where RAVE, through its interaction with subunit C, is facilitating V(1) peripheral stalk subunit interactions with V(0) during V-ATPase assembly.

HCN2 and HCN4 Isoforms Self-assemble and Co-assemble with Equal Preference to Form Functional Pacemaker Channels

Hyperpolarization-activated cyclic nucleotide-modulated (HCN) "pacemaker" channel subunits are integral membrane proteins that assemble as tetramers to form channels in cardiac conduction tissue and nerve cells. Previous studies have suggested that the HCN2 and HCN4 channel isoforms physically interact when overexpressed in mammalian cells, but whether they are able to co-assemble and form functional channels remains unclear. The extent to which co-assembly occurs over self-assembly and whether HCN2-HCN4 heteromeric channels are formed in native tissue are not known. In this study, we show co-assembly of HCN2 and HCN4 in live Chinese hamster ovary cells using bioluminescence resonance energy transfer (BRET(2)), a novel approach for studying tetramerization of ion channel subunits. Together with results from electrophysiological and imaging approaches, the BRET(2) data show that HCN2 and HCN4 subunits self-assemble and co-assemble with equal preference. We also demonstrate colocalization of HCN2 and HCN4 and a positive correlation of their intensities in the embryonic mouse heart using immunohistochemistry, as well as physical interactions between these isoforms in the rat thalamus by coimmunoprecipitation. Together, these data support the formation of HCN2-HCN4 heteromeric channels in native tissue.

A Novel Role of Brg1 in the Regulation of SRF/MRTFA-dependent Smooth Muscle-specific Gene Expression

Serum response factor (SRF) is a key regulator of smooth muscle differentiation, proliferation, and migration. Myocardin-related transcription factor A (MRTFA) is a co-activator of SRF that can induce expression of SRF-dependent, smooth muscle-specific genes and actin/Rho-dependent genes, but not MAPK-regulated growth response genes. How MRTFA and SRF discriminate between these sets of target genes is still unclear. We hypothesized that SWI/SNF ATP-dependent chromatin remodeling complexes, containing Brahma-related gene 1 (Brg1) or Brahma (Brm), may play a role in this process. Results from Western blotting and qRT-PCR analysis demonstrated that dominant negative Brg1 blocked the ability of MRTFA to induce expression of smooth muscle-specific genes, but not actin/Rho-dependent early response genes, in fibroblasts. In addition, dominant negative Brg1 attenuated expression of smooth muscle-specific genes in primary cultures of smooth muscle cells. MRTFA overexpression did not induce expression of smooth muscle-specific genes in SW13 cells, which lack endogenous Brg1 or Brm. Reintroduction of Brg1 or Brm into SW13 cells restored their responsiveness to MRTFA. Immunoprecipitation assays revealed that Brg1, SRF, and MRTFA form a complex in vivo, and Brg1 directly binds MRTFA, but not SRF, in vitro. Results from chromatin immunoprecipitation assays demonstrated that dominant negative Brg1 significantly attenuated the ability of MRTFA to increase SRF binding to the promoters of smooth muscle-specific genes, but not early response genes. Together these data suggest that Brg1/Brm containing SWI/SNF complexes play a critical role in regulating expression of SRF/MRTFA-dependent smooth muscle-specific genes but not SRF/MRTFA-dependent early response genes.

Characterization of baculovirus constructs lacking either the Ac 101, Ac 142, or the Ac 144 open reading frame

To investigate the role of the gene products encoded from the open reading frames 101, 142, and 144 of Autographa californica multiple nucleopolyhedrovirus (AcMNPV), a set of bacmid knockout and repair constructs were generated. The repair genes were engineered to contain an HA epitope tag at their C-termini. The results of transfection–infection assays and growth curve analyses showed that the Ac 101, 142, and 144 genes were required for infectious virus production. To better characterize the role of these genes in the baculovirus replication cycle, quantitative DNA replication assays were performed and demonstrated that in cells transfected with the Ac 101, 142, or 144 knockouts, DNA replicated with similar kinetics as a control virus. Western blot analyses of budded virus from cells infected with the repair viruses showed that these proteins are associated with the viral nucleocapsid. Furthermore, immunoelectron microscopy of cells transfected with the knockout bacmids revealed defects in nucleocapsid production for all three constructs. From these results we concluded that the gene products encoded from these open reading frames are essential for virus production and may be involved in DNA processing, packaging, or nucleocapsid morphogenesis.

Post-endocytic Sorting of Calcitonin Receptor-like Receptor and Receptor Activity-modifying Protein 1

Calcitonin receptor-like receptor (CLR) and the receptor activity-modifying protein 1 (RAMP1) comprise a receptor for calcitonin gene-related peptide (CGRP). Although CGRP induces endocytosis of CLR/RAMP1, little is known about post-endocytic sorting of these proteins. We observed that the duration of stimulation with CGRP markedly affected post-endocytic sorting of CLR/RAMP1. In HEK and SK-N-MC cells, transient stimulation (10(-7) M CGRP, 1 h), induced CLR/RAMP1 recycling with similar kinetics (2-6 h), demonstrated by labeling receptors in living cells with antibodies to extracellular epitopes. Recycling of CLR/RAMP1 correlated with resensitization of CGRP-induced increases in [Ca(2+)](i). Cycloheximide did not affect resensitization, but bafilomycin A(1), an inhibitor of vacuolar H(+)-ATPases, abolished resensitization. Recycling CLR and RAMP1 were detected in endosomes containing Rab4a and Rab11a, and expression of GTPase-defective Rab4aS22N and Rab11aS25N inhibited resensitization. After sustained stimulation (10(-7) M CGRP, >2 h), CLR/RAMP1 trafficked to lysosomes. RAMP1 was degraded approximately 4-fold more rapidly than CLR (RAMP1, 45% degradation, 5 h; CLR, 54% degradation, 16 h), determined by Western blotting. Inhibitors of lysosomal, but not proteasomal, proteases prevented degradation. Sustained stimulation did not induce detectable mono- or polyubiquitination of CLR or RAMP1, determined by immunoprecipitation and Western blotting. Moreover, a RAMP1 mutant lacking the only intracellular lysine (RAMP1K142R) internalized and was degraded normally. Thus, after transient stimulation with CGRP, CLR and RAMP1 traffic from endosomes to the plasma membrane, which mediates resensitization. After sustained stimulation, CLR and RAMP1 traffic from endosomes to lysosomes by ubiquitin-independent mechanisms, where they are degraded at different rates.

The Stardust Family Protein MPP7 Forms a Tripartite Complex with LIN7 and DLG1 That Regulates the Stability and Localization of DLG1 to Cell Junctions

MPP7, a previously uncharacterized member of the p55 Stardust family of membrane-associated guanylate kinase (MAGUK) proteins, was found in a tripartite complex with DLG1 and LIN7A or LIN7C. MPP7 dimerizes with all three LIN7 family members (LIN7A, -B, and -C) through interaction of the single L27 domain of LIN7 with the carboxyl-terminal L27 domain of MPP7, thereby stabilizing both proteins. The dimer of MPP7 with LIN7A or LIN7C associates with DLG1 through an interaction requiring the amino-terminal L27 domain of MPP7. The amino-terminal L27 domain of MPP7 is not sufficient for interaction with DLG1 but interacts efficiently only if MPP7 is in a complex with LIN7A or -C. Thus the specificity of interaction of DLG1 with the LIN7-MPP7 complex is determined by L27 interactions with both MPP7 and LIN7. The tripartite complex forms in a ratio of 1:1:1 and localizes to epithelial adherens junctions in a manner dependent upon MPP7. Expression of MPP7 stabilizes DLG1 in an insoluble compartment. Expression of MPP7 deleted of the PDZ or Src homology 3 domain redistributes MPP7, DLG1, and LIN7 out of adherens junctions and into the soluble cytoplasmic fraction without changing the localization of E-cadherin. Thus, the stability and localization of DLG1 to cell-cell junctions are complex functions determined by the expression and association of particular Stardust family members together with particular LIN7 family members.

An Antiproliferative Genetic Screening Identifies a Peptide Aptamer That Targets Calcineurin and Up-regulates Its Activity

Peptide aptamers are combinatorial recognition molecules that consist of a constant scaffold protein displaying a doubly constrained variable peptide loop. They bind specifically target proteins and interfere with their function. We have built a peptide aptamer library in a lentiviral expression system to isolate aptamers that inhibit cell proliferation in vitro. Using one of the isolated aptamers (R5G42) as a bait protein, we have performed yeast two-hybrid screening of cDNA libraries and identified calcineurin A as a target protein candidate. R5G42 bound calcineurin A in vitro and stimulated its phosphatase activity. When expressed transiently in human cells, R5G42 induced the dephosphorylation of BAD. We have identified an antiproliferative peptide aptamer that binds calcineurin and stimulates its activity. The use of this ligand may help elucidate the still elusive structural mechanisms of activation and inhibition of calcineurin. Our work illustrates the power of phenotypic screening of combinatorial protein libraries to interrogate the proteome and chart molecular regulatory networks.

Inhibitory Gli3 Activity Negatively Regulates Wnt/β-Catenin Signaling

The Hedgehog (Hh) and Wingless (Wnt) families of secreted signaling molecules have key roles in embryonic development and adult tissue homeostasis [1-3]. In the developing neural tube, Wnt and Shh, emanating from dorsal and ventral regions, respectively, have been proposed to govern the proliferation and survival of neural progenitors [4-10]. Surprisingly, Shh is required for the growth and survival of cells in both ventral and dorsal neural tube [11]. Here we demonstrate that inhibition of Shh signaling causes a reduction in Wnt-mediated transcriptional activation. This reduction requires Gli3. Assays in embryos and cell lines indicate that repressor forms of the Hh-regulated transcription factor, Gli3 (Gli3R), which are generated in the absence of Hh signaling, inhibit canonical Wnt signaling. Gli3R acts by antagonizing active forms of the Wnt transcriptional effector, beta-catenin. Consistent with this, Gli3R appears to physically interact with the carboxy-terminal domain of beta-catenin, a region that includes the transactivation domain. These data offer an explanation for the proliferative defects in Shh null embryos and suggest a novel mechanism for crosstalk between the Hh and Wnt pathways.

The construction of the eukaryotic expression plasmid pcDNA3.1/azurin and the increased apoptosis of U2OS cells transfected with it

In our previous study, we demonstrated that azurin could selectively trigger apoptosis in human osteosarcoma cell line U2OS cells. However, the rate of apoptosis (35.8 ± 3.2%) is not very high, and azurin is too expensive to obtain readily. To solve these problems, we constructed a eukaryotic expression plasmid containing the azurin gene with an influenza virus haemagglutinin 9 peptide HA epitope tag, and transfected the recombinant plasmid pcDNA3.1(+)/azurin into U2OS cells. RT-PCR and Western blot analysis validated the successful transfection and the expression of the azurin-HA protein. Conspicuous apoptosis of the transfected cells was detected by flow cytometry (FCM) and the DNA ladder test. The apoptosis rate reached 64.3 ± 13.1%. The transcriptional levels of the Bax and p53 genes increased significantly in U2OS cells transfected with pcDNA3.1(+)/azurin, but the Bcl-2 mRNA level decreased. There was no difference in the levels of Bcl-xl mRNA and Survivin mRNA. We propose that the transfection of the recombinant plasmid pcDNA3.1(+)/azurin can significantly induce apoptosis in U2OS cells. This is closely associated with the up-regulation of the transcriptional level of the Bax and p53 genes, and the down-regulation of that of the Bcl-2 gene.

The Src Homology 3 Domain of the β-Subunit of Voltage-gated Calcium Channels Promotes Endocytosis via Dynamin Interaction

High voltage-gated calcium channels enable calcium entry into cells in response to membrane depolarization. Association of the auxiliary beta-subunit to the alpha-interaction-domain in the pore-forming alpha1-subunit is required to form functional channels. The beta-subunit belongs to the membrane-associated guanylate kinase class of scaffolding proteins containing a Src homology 3 and a guanylate kinase domain. Although the latter is responsible for the high affinity binding to the alpha-interaction domain, the functional significance of the Src homology 3 domain remains elusive. Here, we show that injection of isolated beta-subunit Src homology 3 domain into Xenopus laevis oocytes expressing the alpha1-subunit reduces the number of channels in the plasma membrane. This effect is reverted by coexpressing alpha1 with a dominant-negative mutant of dynamin, a GTPase involved in receptor-mediated endocytosis. Full-length beta-subunit also down-regulates voltage-gated calcium channels but only when lacking the alpha-interaction domain. Moreover, isolated Src homology 3 domain and the full-length beta-subunit were found to interact in vitro with dynamin and to internalize the distantly related Shaker potassium channel. These results demonstrate that the beta-subunit regulates the turnover of voltage-gated calcium channels and other proteins in the cell membrane. This effect is mediated by dynamin and depends on the association state of the beta-subunit to the alpha1-pore-forming subunit. Our findings define a novel function for the beta-subunit through its Src homology 3 domain and establish a link between voltage-gated calcium channel activity and the cell endocytic machinery.

Interruption of CryAB-Amyloid Oligomer Formation by HSP22

An R120G missense mutation in alpha-B-crystallin (CryAB), a small heat-shock protein (HSP), causes a desmin-related cardiomyopathy (DRM) that is characterized by the formation of aggregates containing CryAB and desmin. The mutant CryAB protein leads to the formation of inclusion bodies, which contain amyloid oligomer intermediates (amyloid oligomer) in the cardiomyocytes. To further address the underlying mechanism(s) of amyloid oligomer formation in DRM linked to the CryAB R120G, a recombinant CryAB R120G protein was generated. The purified CryAB R120G protein can form a toxic amyloid oligomer, whereas little immunoreactivity was observed in the wild-type CryAB protein. A native PAGE showed that the oligomerized form was present in the CryAB R120G protein, whereas only a high molecular mass was detected in the wildtype CryAB. The oligomerized CryAB R120G of around 240-480 kDa showed strong positive immunoreactivity against an anti-oligomer antibody. The CryAB R120G amyloid oligomer was unstable and easily lost its conformation by beta-mercaptoethanol and SDS. Recombinant HSP25 or HSP22 proteins can directly interrupt oligomer formation by the CryAB R120G protein, whereas the amyloid oligomer is still present in the mixture of the wild-type CryAB and CryAB R120G proteins. This interruption by HSP25 and HSP22 was confirmed in a cardiomyocyte-based study using an adenoviral transfection system. Blockade of amyloid oligomer formation by HSP25 and HSP22 recovered the ubiquitin proteosomal activity and cellular viability. Blockade of oligomer formation by small HSP may be a new therapeutic strategy for treating DRM as well as other types of amyloid-based degenerative diseases.