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ABSTRACT: Candidate interactions identified by high-throughput protein microarray screening require rigorous -confirmation. Such validation is time-consuming and labor-intensive using conventional techniques. We describe a medium-throughput validation protocol based on coprecipitation of biotin-labeled -proteins synthesized in vitro using a rabbit reticulocyte lysate-coupled transcription and translation system. As our experimental system is based on screening for serum antibodies, we also present methods on purifying immunoglobulin from serum and quantifying the amount of coprecipitated (immunoprecipitated) target protein on Western blot. This technique provides a sensitive confirmatory test allowing for the rapid elimination of false positives prior to more extensive validation and analysis of target interactions in their native environment.
Methods in molecular biology (Clifton, N.J.) 01/2011; 723:239-54.
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Ovidiu Marina,
Ursula Hainz,
Melinda A Biernacki,
Wandi Zhang,
Ann Cai, Jonathan S Duke-Cohan,
Fenglong Liu,
Vladimir Brusic,
Donna Neuberg,
Jeffery L Kutok,
Edwin P Alyea,
Christine M Canning,
Robert J Soiffer,
Jerome Ritz,
Catherine J Wu
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ABSTRACT: Patients with chronic lymphocytic leukemia (CLL) who relapse after allogeneic transplant may achieve durable remission following donor lymphocyte infusion (DLI), showing the potency of donor-derived immunity in eradicating tumors. We sought to elucidate the antigenic basis of the effective graft-versus-leukemia (GvL) responses associated with DLI for the treatment of CLL by analyzing the specificity of plasma antibody responses developing in two DLI-treated patients who achieved long-term remission without graft-versus-host disease. By probing high-density protein microarrays with patient plasma, we discovered 35 predominantly intracellular antigens that elicited high-titer antibody reactivity greater in post-DLI than in pre-DLI plasma. Three antigens-C6orf130, MDS032, and ZFYVE19-were identified by both patients. Along with additional candidate antigens DAPK3, SERBP1, and OGFOD1, these proteins showed higher transcript and protein expression in B cells and CLL cells compared with normal peripheral blood mononuclear cells. DAPK3 and the shared antigens do not represent minor histocompatibility antigens, as their sequences are identical in both donor and tumor. Although ZFYVE19, DAPK3, and OGFOD1 elicited minimal antibody reactivity in 12 normal subjects and 12 chemotherapy-treated CLL patients, 5 of 12 CLL patients with clinical GvL responses were serologically reactive to these antigens. Moreover, antibody reactivity against these antigens was temporally correlated with clinical disease regression. These B-cell antigens represent promising biomarkers of effective anti-CLL immunity.
Cancer Research 02/2010; 70(4):1344-55. · 7.86 Impact Factor
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ABSTRACT: The lipid phosphatase PTEN functions as a tumor suppressor by dephosphorylating the D3 position of phosphoinositide-3,4,5-trisphosphate, thereby negatively regulating the phosphoinositide 3-kinase (PI3K)/AKT signaling pathway. In mammalian cells, PTEN exists either as a monomer or as a part of a >600-kDa complex (the PTEN-associated complex [PAC]). Previous studies suggest that the antagonism of PI3K/AKT signaling by PTEN may be mediated by a nonphosphorylated form of the protein resident within the multiprotein complex. Here we show that PTEN associates with p85, the regulatory subunit of PI3K. Using newly generated antibodies, we demonstrate that this PTEN-p85 association involves the unphosphorylated form of PTEN engaged within the PAC and also includes the p110beta isoform of PI3K. The PTEN-p85 association is enhanced by trastuzumab treatment and linked to a decline in AKT phosphorylation in some ERBB2-amplified breast cancer cell lines. Together, these results suggest that integration of p85 into the PAC may provide a novel means of downregulating the PI3K/AKT pathway.
Molecular and cellular biology 07/2009; 29(19):5377-88. · 6.06 Impact Factor
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ABSTRACT: Modified Vaccinia Ankara (MVA) is a replication-defective strain of vaccinia virus (VV) that is being investigated in humans as an alternative vaccine against smallpox. Understanding the parameters of a MVA vaccine regimen that can effectively enhance protective immunity will be important for clinical development. The present studies utilize cohorts of rhesus monkeys immunized with recombinant MVA (rMVA) or recombinant VV (rVV) vaccine vectors to investigate the magnitude, breadth, and durability of anti-VV immunity elicited by a single or multi-dose vaccine regimen. These data demonstrate that a single immunization with rMVA elicits weaker cellular and humoral immunity compared to a single inoculation with rVV. However, vaccine-elicited antibody responses, but not T cell responses, are significantly enhanced with repeated immunizations of rMVA. Importantly, only monkeys receiving up to four inoculations with rMVA generated neutralizing antibody (NAb) responses that were comparable in magnitude and durability to those elicited in monkeys receiving two inoculations with rVV. These data also show that the breadth of antibody responses against protein antigens associated with two antigenically distinct forms of infectious VV are similar in rMVA- and rVV-immunized monkeys. Together, these studies suggest that a multi-dose vaccine regimen utilizing up to four inoculations of MVA generates robust and durable antibody-mediated immunity comparable to that elicited by replication-competent VV.
Vaccine 02/2009; 27(10):1549-56. · 3.77 Impact Factor
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ABSTRACT: The renewed interest in strategies to combat infectious agents with epidemic potential has led to a re-examination of vaccination protocols against smallpox. To help define which antigens elicit a human antibody response, we have targeted proteins known or predicted to be presented on the surface of the intracellular mature virion (IMV) or the extracellular enveloped virion (EEV). The predicted ectodomains were expressed in a mammalian in vitro coupled transcription/translation reaction using tRNA(lys) precharged with lysine-epsilon-biotin followed by solid phase immobilization on 384-well neutravidin-coated plates. The generated array is highly specific and sensitive in a micro-ELISA format. By comparison of binding of vaccinia-immune sera to the reticulocyte lysate-produced proteins and to secreted post-translationally modified proteins, we demonstrate that for several proteins including the EEV proteins B5 and A33, proper recognition is dependent upon appropriate folding, with little dependence upon glycosylation per se. We further demonstrate that the humoral immune response to vaccinia among different individuals is not uniform in specificity or strength, as different IMV and EEV targets predominate within the group of immunogenic proteins. This heterogeneity likely results from the diversity of HLA Class II alleles and CD4 T helper cell epitopes stimulating B cell antibody production. Our findings have important implications both for design of new recombinant subunit vaccines as well as for methods of assaying the human antibody response utilizing recombinant proteins produced in vitro.
Vaccine 02/2009; 27(8):1154-65. · 3.77 Impact Factor
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ABSTRACT: Precise intrathymic cell migration is important for thymocyte maturation and organ architecture. The orchestration of thymocyte trafficking, however, is not well understood at a molecular level. Here, we described highly regulated plexinD1 expression on CD4+CD8+ double positive (DP) thymocytes. PlexinD1 expression was further affected by the engagement of T cell receptor complex. Activation of plexinD1 via the ligand, semaphorin 3E, repressed CCL25 chemokine signaling via its receptor CCR9 in CD69+ thymocytes. In the absence of plexinD1, CD69+ thymocytes remained in the cortex, maturing to form ectopic single positive (SP) thymocyte clusters in Plxnd1-deficient fetal liver cell-transplanted mice. As a consequence, the boundary between DP and SP thymocytes at corticomedullary junctions was disrupted and medullary structures formed under the thymic capsule. These results demonstrate the importance of plexinD1 in directing migration of maturing thymocytes via modulation of biological responses to chemokine gradients.
Immunity 12/2008; 29(6):888-98. · 21.64 Impact Factor
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ABSTRACT: Mutations at the attractin (Atrn) locus in mice result in altered pigmentation on an agouti background, higher basal metabolic rate and juvenile-onset hypomyelination leading to neurodegeneration, while studies on human immune cells indicate a chemotaxis regulatory function. The underlying biochemical defect remains elusive. In this report we identify a role for attractin in plasma membrane maintenance. In attractin's absence there is a decline in plasma membrane glycolipid-enriched rafts from normal levels at 8 weeks to a complete absence by 24 weeks. The structural integrity of lipid rafts depends upon cholesterol and sphingomyelin, and can be identified by partitioning within of ganglioside GM(1). Despite a significant fall in cellular cholesterol with maturity, and a lesser fall in both membrane and total cellular GM(1), these parameters lag behind raft loss, and are normal when hypomyelination/neurodegeneration has already begun thus supporting consequence rather than cause. These findings can be recapitulated in Atrn-deficient cell lines propagated in vitro. Further, signal transduction through complex membrane receptor assemblies is not grossly disturbed despite the complete absence of lipid rafts. We find these results compatible with a role for attractin in plasma membrane maintenance and consistent with the proposal that the juvenile-onset hypomyelination and neurodegeneration represent a defect in attractin-mediated raft-dependent myelin biogenesis.
Experimental Cell Research 03/2007; 313(4):761-71. · 3.58 Impact Factor
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Viviana Romero,
Charles E Larsen, Jonathan S Duke-Cohan,
Edward A Fox,
Tatiana Romero,
Olga P Clavijo,
Dolores A Fici,
Zaheed Husain,
Ingrid Almeciga,
Dennis R Alford,
Zuheir L Awdeh,
Joaquin Zuñiga,
Lama El-Dahdah,
Chester A Alper,
Edmond J Yunis
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ABSTRACT: The definition of human MHC class I haplotypes through association of HLA-A, HLA-Cw and HLA-B has been used to analyze ethnicity, population migrations and disease association.
Here, we present HLA-E allele haplotype association and population linkage disequilibrium (LD) analysis within the ~1.3 Mb bounded by HLA-B/Cw and HLA-A to increase the resolution of identified class I haplotypes. Through local breakdown of LD, we inferred ancestral recombination points both upstream and downstream of HLA-E contributing to alternative block structures within previously identified haplotypes. Through single nucleotide polymorphism (SNP) analysis of the MHC region, we also confirmed the essential genetic fixity, previously inferred by MHC allele analysis, of three conserved extended haplotypes (CEHs), and we demonstrated that commercially-available SNP analysis can be used in the MHC to help define CEHs and CEH fragments.
We conclude that to generate high-resolution maps for relating MHC haplotypes to disease susceptibility, both SNP and MHC allele analysis must be conducted as complementary techniques.
BMC Genetics 02/2007; 8:14. · 2.47 Impact Factor
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ABSTRACT: The immune cell adaptor adhesion and degranulation promoting adaptor protein (ADAP) and its binding to T-cell adaptor Src kinase-associated protein of 55 kDa (SKAP-55) play a key role in the modulation of T-cell adhesion. While primary binding occurs via SKAP-55 SH3 domain binding to a proline-rich region in ADAP, a second interaction occurs between the ADAP C-terminal SH3 domain (ADAP-SH3c) and a non-canonical RKXXY294XXY297 motif in SKAP-55. Increasing numbers of non-canonical SH3 domain binding motifs have been identified in a number of biological systems. The presence of tyrosine residues in the SKAP-55 RKXXY294XXY297 motif suggested that phosphorylation might influence this unusual SH3 domain interaction. Here, we show that the Src kinase p59fyn can induce the in vivo phosphorylation of the motif, and this event blocks ADAP-SH3c domain binding to the peptide motif. The importance of tyrosine phosphorylation was confirmed by plasmon resonance interaction analysis showing that phosphorylation of Tyr294 residue plays a central role in mediating dissociation, whereas phosphorylation of the second Tyr297 had no effect. Although loss of this secondary interaction did not result in the disruption of the complex, the Y294F mutation blocked T-cell receptor-induced up-regulation of lymphocyte function-associated antigen-1-mediated adhesion to intercellular adhesion molecule-1 and interleukin-2 promoter activity. Our findings identify a RKXXY294 motif in SKAP-55 that mediates unique ADAP SH3c domain binding and is needed for LFA-1-mediated adhesion and cytokine production.
Journal of Biological Chemistry 06/2006; 281(19):13743-50. · 4.77 Impact Factor
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ABSTRACT: First discovered as a circulating secreted molecule expressed by activated T lymphocytes, attractin was examined as a potential marker of immune activity. The discovery that a transmembrane form not only controls neuropeptide regulation of hair pigmentation in animals but also affects basal metabolism led to proposals that attractin may also be an extracellular target amenable for the development of obesity-regulating drugs. Examination of several animal mutants used as models ofjuvenile-onset neurodegeneration revealed mutations at the attractin locus as the cause, and the reassessment of earlier attractin mutants demonstrated that neurodegeneration, alterations in pigmentation regulation, and basal metabolic rate were common to all the allelic variants. The presentation and severity of the symptoms differ depending upon the mutation, and some may be variably penetrant even within an allelic line. In this report, we review our rapidly altering perception of the functional activity of attractin with each further addition to its sphere of physiological involvement. We further reappraise our concepts of the subcellular location of attractin, leading to a new proposal that provides a unifying mechanism for attractin's pleiotropic activities. Progress in elucidating each new aspect of attractin function provides a case study in the evolution of possible therapeutic interventions as well as illuminating some of the pitfalls of studying molecular pathways isolated from the whole organism physiology.
Journal of Environmental Pathology Toxicology and Oncology 02/2004; 23(1):1-11. · 1.00 Impact Factor
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ABSTRACT: Mutations at the Atrn locus that encodes a transmembrane protein with a large ectodomain are responsible for a juvenile-onset neurodegeneration manifest as hypomyelination and cerebral vacuole development in several rodent species. In addition to a membrane isoform, the human Atm locus generates by alternative splicing a secreted form corresponding to the entire ectodomain that then circulates at high concentration in the periphery, released in part by activated T lymphocytes. We report here that the secreted form mRNA is downregulated throughout representative discrete regions of the human brain while membrane attractin mRNA is well represented, resulting in the apparent absence of secreted attractin protein in cerebrospinal fluid (CSF). Transcription of attractin secreted form mRNA is strongly downregulated upon differentiation of a human cortical neuron-derived cell line (HCN-1A) to a mature neuron phenotype in response to nerve growth factor. Recombinant secreted attractin disrupts neurite formation by differentiated HCN-1A cells, resulting in higher levels of branching with shorter processes. This effect is duplicated by anti-attractin and by human serum but not by human serum depleted of attractin or by CSF We propose that inappropriate expression of secreted attractin in the CNS blocks membrane attractin function and that its presence, either by leakage from the periphery, aberrant transcription, or release from inflammatory foci may affect neuron extracellular interactions leading to neurodegeneration in the human.
Journal of Neuropathology and Experimental Neurology 10/2002; 61(9):767-77. · 4.26 Impact Factor
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01/2002; , ISBN: 9780471203070
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ABSTRACT: Src-homology 3 (SH3) domains recognize PXXP core motif preceded or followed by positively charged residue(s). Whether SH3 domains recognize motifs other than proline-based sequences is unclear. In this study, we report SH3 domain binding to a novel proline-independent motif in immune cell adaptor SKAP55, which is comprised of two N-terminal lysine and arginine residues followed by two tyrosines (i.e. RKxxYxxY). Domains capable of binding to class I proline motifs bound to the motif, while the class II domains failed to bind. Peptide precipitation, alanine scanning and in vivo co-expression studies demonstrated a requirement for the arginine, lysine and tandem tyrosines of the motif. Two-dimensional NMR analysis of the peptide bound FYN-SH3 domain showed overlap with the binding site of a proline-rich peptide on the charged surface of the SH3 domain, while resonance signals for other residues (W119, W120, Y137) were not perturbed by the RKGDYASY based peptide. Expression of the RKGDYASY peptide potently inhibited TcRζ/CD3-mediated NF-AT transcription in T cells. Our findings extend the repertoire of SH3 domain binding motifs to include a tyrosine-based motif and demonstrate a regulatory role for this motif in receptor signaling.
