Biomedical subjects

Ettore Appella

Publications and source records attributed to Ettore Appella.

At least 19 recordsLinked to original sources

PubMed2007

Studying multisite binary and ternary protein interactions by global analysis of isothermal titration calorimetry data in SEDPHAT: application to adaptor protein complexes in cell signaling.

Multisite interactions and the formation of ternary or higher-order protein complexes are ubiquitous features of protein interactions. Cooperativity between different ligands is a hallmark for information transfer, and is frequently critical for the biological function. We describe a new computational platform for the global analysis of isothermal titration calorimetry (ITC) data for the study of binary and ternary multisite interactions, implemented as part of the public domain multimethod analysis software SEDPHAT. The global analysis of titrations performed in different orientations was explored, and the potential for unraveling cooperativity parameters in multisite interactions was assessed in theory and experiment. To demonstrate the practical potential and limitations of global analyses of ITC titrations for the study of cooperative multiprotein interactions, we have examined the interactions of three proteins that are critical for signal transduction after T-cell activation, LAT, Grb2, and Sos1. We have shown previously that multivalent interactions between these three molecules promote the assembly of large multiprotein complexes important for T-cell receptor activation. By global analysis of the heats of binding observed in sets of ITC injections in different orientations, which allowed us to follow the formation of binary and ternary complexes, we observed negative and positive cooperativity that may be important to control the pathway of assembly and disassembly of adaptor protein particles.

Adaptor Proteins, Signal Transducing↗

PubMed2006

Regulation of ATM/p53-dependent suppression of myc-induced lymphomas by Wip1 phosphatase.

The ataxia telangiectasia mutated (ATM) kinase is a key tumor suppressor that regulates numerous cell cycle checkpoints as well as apoptosis. Here, we report that ATM is a critical player in the regulation of apoptosis and lymphomagenesis in the presence of c-myc. In turn, deletion of the inhibitory ATM phosphatase, Wip1, results in ATM up-regulation and suppression of Emicro-myc-induced B cell lymphomas. Using mouse genetic crosses, we show that the onset of myc-induced lymphomas is dramatically delayed in Wip1-null mice in an ATM- and p53-, but not p38 MAPK- or Arf-, dependent manner. We propose that Wip1 phosphatase is critical for regulating the ATM-mediated tumor surveillance network.

Animals↗

PubMed2006

The wild-type sequence (wt) p53(25-35) peptide induces HLA-DR7 and HLA-DR11-restricted CD4+ Th cells capable of enhancing the ex vivo expansion and function of anti-wt p53(264-272) peptide CD8+ T cells.

Tumor peptide-based vaccines are more effective when they include tumor-specific Th cell-defined as well as CTL-defined peptides. Presently, two overlapping wild-type sequences (wt) p53 helper peptides, p53(108-122) and p53(110-124), have been identified as HLA-DR1- and/or HLA-DR4-restricted epitopes. These HLA-DR alleles are expressed by approximately 35% of subjects with cancer. To identify Th cell-defined wt p53 peptides suitable for use on the remaining subject population, a dendritic cell (DC)-based coculture system was developed. CD4+ T cells isolated from PBMC obtained from HLA-DR4- normal donors were stimulated ex vivo with autologous DC transfected with wt p53 or mutant p53 cDNA. Reactivity of T cells was tested in ELISPOT IFN-gamma assays against DC pulsed individually with a panel of algorithm-predicted, multiple HLA-DR-binding wt p53 peptides. The wt p53(25-35) peptide was identified as capable of inducing and being recognized by CD4+ T cells in association, at a minimum, with HLA-DR7 and -DR11 molecules, each of which is expressed by approximately 15% of the population. In addition, the presence of anti-p53(25-35) CD4+ Th cells was shown to enhance the in vitro generation/expansion of HLA-A2-restricted, anti-wt p53(264-272) CD8+ T cells, which from one donor were initially "nonresponsive" to the wt p53(264-272) peptide. The wt p53(25-35) peptide has attributes of a naturally presented Th cell-defined peptide, which could be incorporated into antitumor vaccines applicable to a broader population of subjects for whom a wt p53 helper peptide is presently unavailable, as well as used for monitoring anti-p53 Th cell activity in cancer subjects receiving p53-based immunotherapy.

Amino Acid Sequence↗

PubMed2006

Development of a substrate-based cyclic phosphopeptide inhibitor of protein phosphatase 2Cdelta, Wip1.

The wild-type p53-induced phosphatase, Wip1 (PP2Cdelta or PPM1D) is a member of the protein phosphatase 2C (PP2C) family and functions as a negative regulator of the p38 MAP kinase-p53 signaling pathway. PPM1D is amplified or Wip1 is overexpressed in several human cancers, and it acts as a weak oncogene. Although inhibition of Wip1 may have therapeutic value, no specific inhibitors are available. In this study, we designed phosphopeptide inhibitors for Wip1 on the basis of its optimal substrate sequence. We found that phosphoserine-containing diphosphorylated peptides with the sequence pSXpY inhibited Wip1 phosphatase activity, whereas phosphothreonine-containing peptides with the sequence pTXpY were physiological substrates. Moreover, the X residue in the pSXpY sequence modulated inhibitor activity, and beta-branched amino acid-substituted (Ile or Val) phosphopeptides showed high inhibitory potencies. A thioether cyclic phosphopeptide c(MpSIpYVA) had a K(i) <1.0 microM. Two serine/threonine phosphatases, PP2Calpha and PP2A, were not significantly inhibited by the cyclic phosphopeptide with a nonhydrolyzable phosphoserine mimetic. A homology model of Wip1 bound to a cyclic phosphopeptide and site-directed mutagenesis helped to identify residues important for Wip1 inhibitor selectivity among the PP2C family. These results provide the first proof of concept of a specific inhibitor of the catalytic site of Wip1 and should be useful for developing potential anti-cancer drugs.

Amino Acid Sequence↗

PubMed2006

Comparison of the specificity of interaction of cellular and viral zinc-binding domains with 2-mercaptobenzamide thioesters.

The interactions of two 2-mercaptobenzamide thioester compounds with six diverse zinc-binding domains (ZBDs) have been analyzed by UV/visible spectroscopy, NMR spectroscopy, and nucleic acid binding assays. These thioester compounds serve as useful tools for probing the intrinsic chemical stability of ZBDs that exist within a variety of cellular and viral proteins. In our studies, the classical (Cys(2)His(2)) zinc finger ZBDs, the interleaved RING like ZBDs of protein kinase C delta (Cys(2)HisCys and HisCys(3)), and the carboxyl-terminal (Cys(2)HisCys) ZBD of Mouse Mammary Tumor Virus nucleocapsid protein (MMTV NCp10) were resistant to reaction with the thioester compounds. In contrast, the thioester compounds were able to efficiently eject zinc from the amino-terminal (Cys(2)HisCys) ZBD of MMTV NCp10, a Cys(2)HisCys ZBD from Friend of GATA-1 (FOG-1), and from both Cys(4) ZBDs of GATA-1. In all cases, zinc ejection led to a loss of protein structure. Interestingly, GATA-1 was resistant to reaction with the thioester compounds when bound to its target DNA sequence. The electronic and steric screening was calculated for select ZBDs to further explore their reactivity. Based on these results, it appears that both first and second zinc-coordination shell interactions within ZBDs, as well as nucleic acid binding, play important roles in determining the chemical stability and reactivity of ZBDs. These studies not only provide information regarding the relative reactivity of cysteine residues within structural ZBDs but also are crucial for the design of future therapeutic agents that selectively target ZBDs, such as those that occur in the HIV-1 nucleocapsid protein.

Amino Acid Sequence↗

PubMed2006

Wip1 phosphatase modulates ATM-dependent signaling pathways.

Deletion of Ppm1d, the gene encoding the Wip1 phosphatase, renders cells resistant to transformation and mice resistant to tumor development. Here, we report that deficiency of Wip1 resulted in activation of the ataxia-telangiectasia mutated (ATM) kinase. In turn, overexpression of Wip1 was sufficient to reduce activation of the ATM-dependent signaling cascade after DNA damage. Wip1 dephosphorylated ATM Ser1981, a site critical for ATM monomerization and activation, and was critical for resetting ATM phosphorylation as cells repaired damaged DNA. We propose that the Wip1 phosphatase is an integral component of an ATM-dependent signaling pathway.

Animals↗

PubMed2006

Oligomerization of signaling complexes by the multipoint binding of GRB2 to both LAT and SOS1.

Receptor oligomerization is vital for activating intracellular signaling, in part by initiating events that recruit effector and adaptor proteins to sites of active signaling. Whether these distal molecules themselves oligomerize is not well appreciated. In this study, we examined the molecular interactions of the adaptor protein GRB2. In T cells, the SH2 domain of GRB2 binds phosphorylated tyrosines on the adaptor protein LAT and the GRB2 SH3 domains associate with the proline-rich regions of SOS1 and CBL. Using biochemical and biophysical techniques in conjunction with confocal microscopy, we observed that the simultaneous association of GRB2, via its SH2 and SH3 domains, with multivalent ligands led to the oligomerization of these ligands, which affected signaling. These data suggest that multipoint binding of distal adaptor proteins mediates the formation of oligomeric signaling clusters vital for intracellular signaling.

Adaptor Proteins, Signal Transducing↗

PubMed2006

Structure of the Tfb1/p53 complex: Insights into the interaction between the p62/Tfb1 subunit of TFIIH and the activation domain of p53.

The interaction between the amino-terminal transactivation domain (TAD) of p53 and TFIIH is directly correlated with the ability of p53 to activate both transcription initiation and elongation. We have identified a region within the p53 TAD that specifically interacts with the pleckstrin homology (PH) domain of the p62 and Tfb1 subunits of human and yeast TFIIH. We have solved the 3D structure of a complex between the p53 TAD and the PH domain of Tfb1 by NMR spectroscopy. Our structure reveals that p53 forms a nine residue amphipathic alpha helix (residues 47-55) upon binding to Tfb1. In addition, we demonstrate that diphosphorylation of p53 at Ser46 and Thr55 leads to a significant enhancement in p53 binding to p62 and Tfb1. These results indicate that a phosphorylation cascade involving Ser46 and Thr55 of p53 could play an important role in the regulation of select p53 target genes.

Gene Expression Regulation↗

PubMed2006

Wip1 phosphatase-deficient mice exhibit defective T cell maturation due to sustained p53 activation.

The PP2C phosphatase Wip1 dephosphorylates p38 and blocks UV-induced p53 activation in cultured human cells. Although the level of TCR-induced p38 MAPK activity is initially comparable between Wip1-/- and wild-type thymocytes, phosphatase-deficient cells failed to down-regulate p38 MAPK activity after 6 h. Analysis of young Wip1-deficient mice showed that they had fewer splenic T cells. Their thymi were smaller, contained significantly fewer cells, and failed to undergo age-dependent involution compared with wild-type animals. Analysis of thymocyte subset numbers by flow cytometry suggested that cell numbers starting at the double-negative (DN)4 stage are significantly reduced in Wip1-deficient mice, and p53 activity is elevated in cell-sorted DN4 and double-positive subpopulations. Although apoptosis and proliferation was normal in Wip1-/- DN4 cells, they appeared to be in cell cycle arrest. In contrast, a significantly higher percentage of apoptotic cells were found in the double-positive population, and down-regulation of thymocyte p38 MAPK activation by anti-CD3 was delayed. To examine the role of p38 MAPK in early thymic subpopulations, fetal thymic organ cultures cultured in the presence/absence of a p38 MAPK inhibitor did not correct the thymic phenotype. In contrast, the abnormal thymic phenotype of Wip1-deficient mice was reversed in the absence of p53. These data suggest that Wip1 down-regulates p53 activation in the thymus and is required for normal alphabeta T cell development.

Animals↗

PubMed2006

Tumor susceptibility and apoptosis defect in a mouse strain expressing a human p53 transgene.

Activation of apoptosis is believed to be critical for the role of p53 as a tumor suppressor. Here, we report a new mouse strain carrying a human p53 transgene in the mouse p53-null background. Expression of human p53 in these mice was comparable with wild-type murine p53; however, transactivation, induction of apoptosis, and G(1)-S checkpoint, but not transrepression or regulation of a centrosomal checkpoint, were deregulated. Although multiple functions of p53 were abrogated, mice carrying the human p53 transgene did not show early onset of tumors as typically seen for p53-null mice. In contrast, human p53 in the p53-null background did not prevent accelerated tumor development after genotoxic or oncogenic stress. Such behavior of human p53 expressed at physiologic levels in transgenic cells could be explained by unexpectedly high binding with Mdm2. By using Nutlin-3a, an inhibitor of the interaction between Mdm2 and p53, we were able to partially reconstitute p53 transactivation and apoptosis in transgenic cells. Our findings indicate that the interaction between p53 and Mdm2 controls p53 transcriptional activity in homeostatic tissues and regulates DNA damage- and oncogene-induced, but not spontaneous, tumorigenesis.

Animals↗

PubMed2006

Sorting of Pmel17 to melanosomes through the plasma membrane by AP1 and AP2: evidence for the polarized nature of melanocytes.

Adaptor proteins (AP) play important roles in the sorting of proteins from the trans-Golgi network, but how they function in the sorting of various melanosome-specific proteins such as Pmel17, an essential structural component of melanosomes, in melanocytes is unknown. We characterized the processing and trafficking of Pmel17 via adaptor protein complexes within melanocytic cells. Proteomics analysis detected Pmel17, AP1 and AP2, but not AP3 or AP4 in early melanosomes. Real-time PCR, immunolabeling and tissue in-situ hybridization confirmed the coexpression of AP1 isoforms mu1A and mu1B (expressed only in polarized cells) in melanocytes and keratinocytes, but expression of mu1B is missing in some melanoma cell lines. Transfection with AP1 isoforms (mu1A or mu1B) showed two distinct distribution patterns that involved Pmel17, and only mu1B was able to restore the sorting of Pmel17 to the plasma membrane in cells lacking mu1B expression. Finally, we established that expression of mu1B is regulated physiologically in melanocytes by UV radiation or DKK1. These results show that Pmel17 is sorted to melanosomes by various intracellular routes, directly or indirectly through the plasma membrane, and the presence of basolateral elements in melanocytes suggests their polarized nature.

Adaptor Protein Complex 1↗

PubMed2006

Unfolding, aggregation, and amyloid formation by the tetramerization domain from mutant p53 associated with lung cancer.

The p53 tumor suppressor is a tetrameric transcriptional enhancer, and its activity is compromised by mutations that cause amino acid substitutions in its tetramerization domain. Here we analyze the biochemical and biophysical properties of peptides corresponding to amino acids 319-358 of wild-type human p53, which includes the tetramerization domain, and that of a cancer-derived mutant with valine substituted for glycine 334. Unlike the wild-type peptide, the G334V peptide forms amyloid fibrils by a two-step process under physiological conditions of temperature and pH. Nevertheless, the G334V peptide is capable of forming heterooligomers with a wild-type peptide. Computational modeling of the G334V peptide structure suggests that substitution of valine for glycine 334 causes a local distortion that contributes to a beta-dominated structural transition leading to amyloid formation. Since the distortion is mostly on the surface, the mutant peptide is still able to form a pseudonative tetramer complex at higher concentrations and/or lower temperatures. Our study suggests a new potential mechanism by which mutations that compromise tetramer formation inactivate p53 as a tumor suppressor.

Amino Acid Sequence↗

PubMed2006

Disruption of estrogen receptor DNA-binding domain and related intramolecular communication restores tamoxifen sensitivity in resistant breast cancer.

A serious obstacle to successful treatment of estrogen receptor (ER)-positive human breast cancer is cell resistance to tamoxifen (TAM) therapy. Here we show that the electrophile disulfide benzamide (DIBA), an ER zinc finger inhibitor, blocks ligand-dependent and -independent cell growth of TAM-resistant breast cancer in vitro and in vivo. Such inhibition depends on targeting disruption of the ER DNA-binding domain and its communication with neighboring functional domains, facilitating ERalpha dissociation from its coactivator AIB1 and concomitant association with its corepressor NCoR bound to chromatin. DIBA does not affect phosphorylation of HER2, MAPK, AKT, and AIB1, suggesting that DIBA-modified ERalpha may induce a switch from agonistic to antagonistic effects of TAM on resistant breast cancer cells.

Animals↗

PubMed2006

Proteomic and bioinformatic characterization of the biogenesis and function of melanosomes.

Melanin, which is responsible for virtually all visible skin, hair, and eye pigmentation in humans, is synthesized, deposited, and distributed in subcellular organelles termed melanosomes. A comprehensive determination of the protein composition of this organelle has been obstructed by the melanin present. Here, we report a novel method of removing melanin that includes in-solution digestion and immobilized metal affinity chromatography (IMAC). Together with in-gel digestion, this method has allowed us to characterize melanosome proteomes at various developmental stages by tandem mass spectrometry. Comparative profiling and functional characterization of the melanosome proteomes identified approximately 1500 proteins in melanosomes of all stages, with approximately 600 in any given stage. These proteins include 16 homologous to mouse coat color genes and many associated with human pigmentary diseases. Approximately 100 proteins shared by melanosomes from pigmented and nonpigmented melanocytes define the essential melanosome proteome. Proteins validated by confirming their intracellular localization include PEDF (pigment-epithelium derived factor) and SLC24A5 (sodium/potassium/calcium exchanger 5, NCKX5). The sharing of proteins between melanosomes and other lysosome-related organelles suggests a common evolutionary origin. This work represents a model for the study of the biogenesis of lysosome-related organelles.

Animals↗

PubMed2006

Acetylation of mouse p53 at lysine 317 negatively regulates p53 apoptotic activities after DNA damage.

Posttranslational modifications of p53, including phosphorylation and acetylation, play important roles in regulating p53 stability and activity. Mouse p53 is acetylated at lysine 317 by PCAF and at multiple lysine residues at the extreme carboxyl terminus by CBP/p300 in response to genotoxic and some nongenotoxic stresses. To determine the physiological roles of p53 acetylation at lysine 317, we introduced a Lys317-to-Arg (K317R) missense mutation into the endogenous p53 gene of mice. p53 protein accumulates to normal levels in p53(K317R) mouse embryonic fibroblasts (MEFs) and thymocytes after DNA damage. While p53-dependent gene expression is largely normal in p53(K317R) MEFs after various types of DNA damage, increased p53-dependent apoptosis was observed in p53(K317R) thymocytes, epithelial cells from the small intestine, and cells from the retina after ionizing radiation (IR) as well as in E1A/Ras-expressing MEFs after doxorubicin treatment. Consistent with these findings, p53-dependent expression of several proapoptotic genes was significantly increased in p53(K317R) thymocytes after IR. These findings demonstrate that acetylation at lysine 317 negatively regulates p53 apoptotic activities after DNA damage.

Acetylation↗

PubMed2006

Preclinical evaluation of a zinc finger inhibitor targeting lentivirus nucleocapsid protein in SIV-infected monkeys.

There is a continued need to develop inexpensive and effective drugs specific for novel targets of human immunodeficiency virus type 1 (HIV-1). The HIV-1 nucleocapsid p7 (NCp7) protein plays a critical role in early and late stages of the virus life cycle and possesses two highly conserved retroviral zinc fingers that are essential for its function. We have previously shown that zinc finger inhibitors (ZFI) based on the S-acyl 2-mercaptobenzamide thioester (SAMT) chemotype specifically target HIV NCp7 and are effective at reducing levels of infectious virus in an HIV-1-transgenic mouse model. Here, we did an initial proof-of-concept study to test the potential of a lead SAMT compound to reduce virus infectivity in the simian immunodeficiency virus (SIV) nonhuman primate model. SAMT-19 had potent antiviral and virucidal effects against the primary pathogenic isolate SIV/DeltaB670 and was non-cytotoxic in vitro. Cynomolgus macaques were infected intrarectally with SIV/DeltaB670 and treated with a low dose of SAMT-19 by continuous infusion from day 8 to day 28 post infection. Monkeys in the treatment group had significantly lower levels of infectious virus in peripheral blood mononuclear cells during the course of therapy as compared to monkeys in the control group, although therapy had no demonstrable effect on virus load. SAMT-19 therapy did not alter liver, kidney or immunologic function and was well tolerated by all treated monkeys. These data demonstrate that SAMT-19 is safe and virucidal in the nonhuman primate model. Further studies directed at optimizing SAMT bioavailability and pharmacokinetics likely will result in enhanced therapeutic efficacy of this promising HIV therapeutic.

Animals↗

PubMed2005

Charge modification at multiple C-terminal lysine residues regulates p53 oligomerization and its nucleus-cytoplasm trafficking.

The basal level of the tumor suppressor p53 is regulated by MDM2-mediated ubiquitination at specific lysines, which leads to p53 nuclear export and degradation. Upon p53 activation, however, these lysines become acetylated by p300/CREB-binding protein. Here we have reported an unexpected finding that p300-mediated acetylation also regulates p53 subcellular localization and can promote cytoplasmic localization of p53. This activity is independent of MDM2 but requires a p53 nuclear export signal and acetylation of multiple lysines by p300. Mechanistically, we showed that conversion of a minimal four of these lysines to alanines but not arginines mimics p300-mediated p53 nuclear export, and these lysine-neutralizing mutations effectively prevent p53 tetramerization, thus exposing the oligomerization-regulated nuclear export signal. Our study suggested a threshold mechanism whereby the degree of acetylation regulates p53 nucleus-cytoplasm trafficking by neutralizing a lysine-dependent charge patch, which in turn, controls oligomerization-dependent p53 nuclear export.

Acetylation↗

PubMed2005

A CD8+ T cell heptaepitope minigene vaccine induces protective immunity against Chlamydia pneumoniae.

An intact T cell compartment and IFN-gamma signaling are required for protective immunity against Chlamydia. In the mouse model of Chlamydia pneumoniae (Cpn) infection, this immunity is critically dependent on CD8(+) T cells. Recently we reported that Cpn-infected mice generate an MHC class I-restricted CD8(+) Tc1 response against various Cpn Ags, and that CD8(+) CTL to multiple epitopes inhibit Cpn growth in vitro. Here, we engineered a DNA minigene encoding seven H-2(b)-restricted Cpn CTL epitopes, the universal pan-DR epitope Th epitope, and an endoplasmic reticulum-translocating signal sequence. Immunization of C57BL/6 mice with this construct primed IFN-gamma-producing CD8(+) CTL against all seven CTL epitopes. CD8(+) T cell lines generated to minigene-encoded CTL epitopes secreted IFN-gamma and TNF-alpha and exhibited CTL activity upon recognition of Cpn-infected macrophages. Following intranasal challenge with Cpn, a 3.6 log reduction in mean lung bacterial numbers compared with control animals was obtained. Using a 20-fold increase in the Cpn challenging dose, minigene-vaccinated mice had a 60-fold reduction in lung bacterial loads, compared with controls. Immunization and challenge studies with beta(2)-microglobulin(-/-) mice indicated that the reduction of lung Cpn burdens was mediated by the MHC class I-dependent CD8(+) T cells to minigene-included Cpn CTL epitopes, rather than by pan-DR epitope-specific CD4(+) T cells. This constitutes the first demonstration of significant protection achieved by immunization with a CD8(+) T cell epitope-based DNA construct in a bacterial system and provides the basis for the optimal design of multicomponent anti-Cpn vaccines for humans.

Amino Acid Sequence↗