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1.
PLoS One ; 13(10): e0205756, 2018.
Article in English | MEDLINE | ID: mdl-30335821

ABSTRACT

As a step toward the development of variant forms of Env with enhanced immunogenic properties, we have expressed the glycoprotein in the yeast surface display system in a form that can be subjected to random mutagenesis followed by screening for forms with enhanced binding to germline antibodies. To optimize the expression and immunogenicity of the yeast-displayed Env protein, we tested different approaches for cell wall anchoring, expression of gp120 and gp140 Env from different viral strains, the effects of introducing mutations designed to stabilize Env, and the effects of procedures for altering N-linked glycosylation of Env. We find that diverse forms of HIV envelope glycoprotein can be efficiently expressed at the yeast cell surface and that gp140 forms of Env are effectively cleaved by Kex2p, the yeast furin protease homolog. Multiple yeast-displayed gp120 and gp140 proteins are capable of binding to antibodies directed against the V3-variable loop, CD4 binding site, and gp41 membrane-proximal regions, including some antibodies whose binding is known to depend on Env conformation and N-linked glycan. Based on antibody recognition and sensitivity to glycosidases, yeast glycosylation patterns partially mimic high mannose-type N-glycosylation in mammalian cells. However, yeast-displayed Env is not recognized by some anti-Env antibodies sensitive to quaternary structure, suggesting either that the displayed protein exists in a monomeric state or that for these antibodies, yeast glycosylation in certain regions hinders recognition or access. Consistent with studies in other systems, reconstructed predicted unmutated precursors to anti-Env antibodies exhibit little affinity for the yeast-displayed envelope protein.


Subject(s)
AIDS Vaccines/immunology , HIV Antibodies/immunology , HIV-1/immunology , Saccharomyces cerevisiae/virology , Glycosylation , HEK293 Cells , HIV Envelope Protein gp120/genetics , HIV Envelope Protein gp120/immunology , HIV Envelope Protein gp120/metabolism , HIV-1/genetics , HIV-1/metabolism , Humans , Immunogenicity, Vaccine/genetics , Immunogenicity, Vaccine/immunology , Immunologic Techniques/methods , Mutagenesis, Site-Directed , Mutation , Proprotein Convertases/metabolism , Protein Binding/immunology , Saccharomyces cerevisiae/metabolism , Saccharomyces cerevisiae Proteins/metabolism , env Gene Products, Human Immunodeficiency Virus/genetics , env Gene Products, Human Immunodeficiency Virus/immunology , env Gene Products, Human Immunodeficiency Virus/metabolism
2.
Nanoscale ; 7(47): 20042-54, 2015 Dec 21.
Article in English | MEDLINE | ID: mdl-26568258

ABSTRACT

The increasing use of nanoparticles (NPs) in technological applications and in commercial products has escalated environmental health and safety concerns. The detection of NPs in the environment and in biological systems is challenged by limitations associated with commonly used analytical techniques. In this paper we report on the development and characterization of NP binding antibodies, termed NProbes. Phage display methodology was used to discover antibodies that bind NPs dispersed in solution. We present a proof-of-concept for the generation of NProbes and their use for detecting quantum dots and titanium dioxide NPs in vitro and in an ex vivo human skin model. Continued development and refinement of NProbes to detect NPs that vary in composition, shape, size, and surface coating will comprise a powerful tool kit that can be used to advance nanotechnology research particularly in the nanotoxicology and nanotherapeutics fields.


Subject(s)
Indicators and Reagents/chemistry , Nanoparticles/chemistry , Single-Chain Antibodies/chemistry , Albumins/chemistry , Humans , Immunohistochemistry , Microscopy, Electron, Transmission , Nanomedicine/methods , Peptide Library , Quantum Dots/chemistry , Skin/drug effects , Skin/pathology , Titanium/chemistry
3.
J Clin Invest ; 125(3): 1299-310, 2015 Mar 02.
Article in English | MEDLINE | ID: mdl-25689261

ABSTRACT

Disturbed blood flow (d-flow) causes endothelial cell (EC) dysfunction, leading to atherosclerotic plaque formation. We have previously shown that d-flow increases SUMOylation of p53 and ERK5 through downregulation of sentrin/SUMO-specific protease 2 (SENP2) function; however, it is not known how SENP2 itself is regulated by d-flow. Here, we determined that d-flow activated the serine/threonine kinase p90RSK, which subsequently phosphorylated threonine 368 (T368) of SENP2. T368 phosphorylation promoted nuclear export of SENP2, leading to downregulation of eNOS expression and upregulation of proinflammatory adhesion molecule expression and apoptosis. In an LDLR-deficient murine model of atherosclerosis, EC-specific overexpression of p90RSK increased EC dysfunction and lipid accumulation in the aorta compared with control animals; however, these pathologic changes were not observed in atherosclerotic mice overexpressing dominant negative p90RSK (DN-p90RSK). Moreover, depletion of SENP2 in these mice abolished the protective effect of DN-p90RSK overexpression. We propose that p90RSK-mediated SENP2-T368 phosphorylation is a master switch in d-flow-induced signaling, leading to EC dysfunction and atherosclerosis.


Subject(s)
Atherosclerosis/enzymology , Cysteine Endopeptidases/physiology , Human Umbilical Vein Endothelial Cells/physiology , Ribosomal Protein S6 Kinases, 90-kDa/metabolism , Sumoylation , Active Transport, Cell Nucleus , Animals , Apoptosis , Atherosclerosis/physiopathology , Carotid Arteries/enzymology , Carotid Arteries/pathology , Cells, Cultured , Enzyme Activation , Female , Humans , Mice, Transgenic , Mitogen-Activated Protein Kinase 7/metabolism , Phosphorylation , Plaque, Atherosclerotic/enzymology , Regional Blood Flow , Tumor Suppressor Protein p53/metabolism , Vasculitis/enzymology
4.
Biochemistry ; 53(16): 2581-93, 2014 Apr 29.
Article in English | MEDLINE | ID: mdl-24735378

ABSTRACT

Genomic regions rich in G residues are prone to adopt G-quadruplex structure. Multiple Sp1-binding motifs arranged in tandem have been suggested to form this structure in promoters of cancer-related genes. Here, we demonstrate that the G-rich proviral DNA sequence of the HIV-1 U3 region, which serves as a promoter of viral transcription, adopts a G-quadruplex structure. The sequence contains three binding elements for transcription factor Sp1, which is involved in the regulation of HIV-1 latency, reactivation, and high-level virus expression. We show that the three Sp1 binding motifs can adopt different forms of G-quadruplex structure and that the Sp1 protein can recognize and bind to its site folded into a G-quadruplex. In addition, a c-kit2 specific antibody, designated hf2, binds to two different G-quadruplexes formed in Sp1 sites. Since U3 is encoded at both viral genomic ends, the G-rich sequence is also present in the RNA genome. We demonstrate that the RNA sequence of U3 forms dimers with characteristics known for intermolecular G-quadruplexes. Together with previous reports showing G-quadruplex dimers in the gag and cPPT regions, these results suggest that integrity of the two viral genomes is maintained through numerous intermolecular G-quadruplexes formed in different RNA genome locations. Reconstituted reverse transcription shows that the potassium-dependent structure formed in U3 RNA facilitates RT template switching, suggesting that the G-quadruplex contributes to recombination in U3.


Subject(s)
G-Quadruplexes , HIV-1/genetics , RNA, Small Nucleolar/chemistry , Binding Sites , Circular Dichroism , DNA, Single-Stranded/chemistry , DNA, Viral/chemistry , Dimerization , Genome, Viral , Nucleic Acid Conformation , Promoter Regions, Genetic , RNA, Viral/chemistry , Sp1 Transcription Factor/metabolism
5.
Biochemistry ; 52(10): 1802-13, 2013 Mar 12.
Article in English | MEDLINE | ID: mdl-23394681

ABSTRACT

Mimetics of conformational protein epitopes have broad applications but have been difficult to identify using conventional peptide phage display. The 10th type III domain of human fibronectin (FNfn10) has two extended, randomizable surface-exposed loops and might be more amenable to the identification of such mimetics. We therefore selected a library of FNfn10 clones, randomized in both loops (15 residues in all), for binding to monoclonal antibodies (mAbs) that recognize the HIV-1 envelope glycoprotein. Anti-idiotypic monobodies (αIMs) mimicking both "linear" epitopes (2F5 and 4E10 mAbs) and conformational epitopes (b12 and VRC01 mAbs) were generated. αIMs selected against 2F5 and 4E10 frequently displayed sequence homology to the corresponding linear native epitopes. In the case of b12 and VRC01, we expected that the two constrained loop domains of FNfn10 would both contribute to complex conformational interactions with target antibodies. However, mutagenesis studies revealed differences from this simple model. An αIM selected against b12 was found to bind its cognate antibody via only a few residues within the BC loop of FNfn10, with minimal contribution from the FG loop. Unexpectedly, this was sufficient to generate a protein that engaged its cognate antibody in a manner very similar to that of HIV-1 Env, and with a strong KD (43 nM). In contrast, an αIM selected against VRC01 engaged its cognate antibody in a manner that was dependent on both BC and FG loop sequences. Overall, these data suggest that the FNfn10 scaffold can be used to identify complex structures that mimic conformational protein epitopes.


Subject(s)
Antibodies, Anti-Idiotypic/chemistry , Antibodies, Monoclonal/chemistry , Fibronectins/chemistry , Amino Acid Sequence , Antibodies, Anti-Idiotypic/genetics , Antibodies, Anti-Idiotypic/immunology , Antibodies, Monoclonal/genetics , Antibodies, Monoclonal/immunology , Binding, Competitive , Biomimetic Materials/chemistry , Broadly Neutralizing Antibodies , Fibronectins/genetics , HIV Antibodies/blood , HIV Antibodies/chemistry , HIV Antibodies/immunology , HIV-1/chemistry , HIV-1/immunology , Humans , Models, Molecular , Mutagenesis, Site-Directed , Peptide Library , Protein Conformation
6.
Arterioscler Thromb Vasc Biol ; 33(2): 321-9, 2013 Feb.
Article in English | MEDLINE | ID: mdl-23202365

ABSTRACT

OBJECTIVE: Protein inhibitor of activated signal transducer and activator of transcription-1 (PIAS1) is known to function as small ubiquitin-like modifier (SUMO) E3 ligase as well as transrepressor. The aim of the study is to elucidate the regulatory mechanisms for these 2 different functions, especially with respect to endothelial inflammation. METHODS AND RESULTS: The mitogen-activated protein kinase (MAPK)-activated protein kinase-2 is a proinflammatory kinase and phosphorylates PIAS1 at the Ser522 residue. Activation of MAPK-activated protein kinase-2 enhances p53-SUMOylation, but a PIAS1 phosphorylation mutant, PIAS1-S522A, abolished this p53-SUMOylation, suggesting a critical role for PIAS1-S522 phosphorylation in its SUMO ligase activity. Because nuclear p53 can inhibit Kruppel-like factor 2 promoter activity, we investigated the roles for PIAS1 phosphorylation and p53-SUMOylation in the Kruppel-like factor 2 and endothelial NO synthase expression. Both MAPK-activated protein kinase-2 and PIAS1 overexpression increased Kruppel-like factor 2 promoter activity and endothelial NO synthase expression, which were inhibited by expressing a p53-SUMOylation defective mutant, p53-K386R, and PIAS1-S522A. PIAS1-S522A also abolished the anti-inflammatory effect of wild-type PIAS1 in vitro and also in vivo, which was examined by leukocyte rolling in microvessels of skin grafts transduced by adenovirus encoding PIAS1-WT or - S522A mutant. CONCLUSIONS: Our study has identified a novel negative feedback regulatory pathway through which MAPK-activated protein kinase-2 limits endothelial inflammation via the PIAS1 S522 phosphorylation-mediated increase in PIAS1 transrepression and SUMO ligase activity.


Subject(s)
Endothelial Cells/enzymology , Inflammation/prevention & control , Intracellular Signaling Peptides and Proteins/metabolism , Protein Inhibitors of Activated STAT/metabolism , Protein Serine-Threonine Kinases/metabolism , Small Ubiquitin-Related Modifier Proteins/metabolism , Ubiquitin-Protein Ligases/metabolism , Animals , Cells, Cultured , Endothelial Cells/immunology , Enzyme Activation , Gene Expression Regulation , Human Umbilical Vein Endothelial Cells/enzymology , Human Umbilical Vein Endothelial Cells/immunology , Humans , Inflammation/enzymology , Inflammation/immunology , Inflammation Mediators/metabolism , Intracellular Signaling Peptides and Proteins/genetics , Kruppel-Like Transcription Factors/metabolism , Leukocyte Rolling , Mice , Mice, Inbred C57BL , NF-kappa B/metabolism , Nitric Oxide Synthase Type III/metabolism , Phosphorylation , Protein Inhibitors of Activated STAT/genetics , Protein Serine-Threonine Kinases/genetics , RNA Interference , Skin/blood supply , Skin Transplantation , Small Ubiquitin-Related Modifier Proteins/genetics , Sumoylation , Time Factors , Transfection , Transplantation, Autologous , Tumor Necrosis Factor-alpha/metabolism , Tumor Suppressor Protein p53/metabolism , Ubiquitin-Protein Ligases/genetics
7.
Circulation ; 127(4): 486-99, 2013 Jan 29.
Article in English | MEDLINE | ID: mdl-23243209

ABSTRACT

BACKGROUND: Diabetes mellitus is a major risk factor for cardiovascular mortality by increasing endothelial cell (EC) dysfunction and subsequently accelerating atherosclerosis. Extracellular-signal regulated kinase 5 (ERK5) is activated by steady laminar flow and regulates EC function by increasing endothelial nitric oxide synthase expression and inhibiting EC inflammation. However, the role and regulatory mechanisms of ERK5 in EC dysfunction and atherosclerosis are poorly understood. Here, we report the critical role of the p90 ribosomal S6 kinase (p90RSK)/ERK5 complex in EC dysfunction in diabetes mellitus and atherosclerosis. METHODS AND RESULTS: Inducible EC-specific ERK5 knockout (ERK5-EKO) mice showed increased leukocyte rolling and impaired vessel reactivity. To examine the role of endothelial ERK5 in atherosclerosis, we used inducible ERK5-EKO-LDLR(-/-) mice and observed increased plaque formation. When activated, p90RSK associated with ERK5, and this association inhibited ERK5 transcriptional activity and upregulated vascular cell adhesion molecule 1 expression. In addition, p90RSK directly phosphorylated ERK5 S496 and reduced endothelial nitric oxide synthase expression. p90RSK activity was increased in diabetic mouse vessels, and fluoromethyl ketone-methoxyethylamine, a specific p90RSK inhibitor, ameliorated EC-leukocyte recruitment and diminished vascular reactivity in diabetic mice. Interestingly, in ERK5-EKO mice, increased leukocyte rolling and impaired vessel reactivity were resistant to the beneficial effects of fluoromethyl ketone-methoxyethylamine, suggesting a critical role for endothelial ERK5 in mediating the salutary effects of fluoromethyl ketone-methoxyethylamine on endothelial dysfunction. Fluoromethyl ketone-methoxyethylamine also inhibited atherosclerosis formation in ApoE(-/-) mice. CONCLUSIONS: Our study highlights the importance of the p90RSK/ERK5 module as a critical mediator of EC dysfunction in diabetes mellitus and atherosclerosis formation, thus revealing a potential new target for therapeutic intervention.


Subject(s)
Atherosclerosis/physiopathology , Diabetic Angiopathies/physiopathology , Mitogen-Activated Protein Kinase 7/genetics , Mitogen-Activated Protein Kinase 7/metabolism , Ribosomal Protein S6 Kinases, 90-kDa/genetics , Ribosomal Protein S6 Kinases, 90-kDa/metabolism , Amino Acid Chloromethyl Ketones/pharmacology , Animals , Atherosclerosis/drug therapy , Atherosclerosis/metabolism , Diabetic Angiopathies/drug therapy , Diabetic Angiopathies/metabolism , Drug Synergism , Glucose/pharmacology , Human Umbilical Vein Endothelial Cells , Humans , Hydrogen Peroxide/pharmacology , Leukocyte Rolling/physiology , Male , Mice , Mice, Inbred C57BL , Mice, Knockout , Mice, Mutant Strains , Nitric Oxide Synthase Type III/metabolism , Oxidants/pharmacology , Phosphorylation/physiology , Rats , Ribosomal Protein S6 Kinases, 90-kDa/antagonists & inhibitors
8.
Methods ; 58(1): 62-8, 2012 Sep.
Article in English | MEDLINE | ID: mdl-22819856

ABSTRACT

A major goal in the study of autoimmune disease is the identification of biomarkers of disease to allow early diagnosis and initiation of treatment. The production of autoantibodies is the key feature of most autoimmune disease, so much effort has focused on characterizing the antigens reactive with these antibodies. However, even for the most well understood autoimmune diseases like rheumatoid arthritis and systemic lupus erythematosus, identification of antigens that detect autoantibodies in all patients have yet to be discovered. We describe a novel strategy for deriving mimotopes to disease-specific serum antibodies by selecting anti-idiotypic monobodies from a large molecular diversity library. Monobodies are derived by partial randomization of two surface exposed loops of a fibronectin domain scaffold in a phage display vector. The phage library is selected for binding to serum antibodies using a subtractive panning strategy. We evaluated this strategy by selecting the monobody library on a pool of serum immunoglobulin derived from a group of rheumatoid arthritis patients and evaluated selected clones for multi-patient reactivity and specificity for rheumatoid arthritis. The use of the fibronectin scaffold to derive stable, easy to produce molecular probes for diagnosis of autoimmune disease could be of significant value in improving diagnostic assays for virtually any disease that exhibits a characteristic immune response.


Subject(s)
Antibodies, Anti-Idiotypic/chemistry , Arthritis, Rheumatoid/blood , Autoantibodies/blood , Cell Surface Display Techniques , Amino Acid Sequence , Antibodies, Anti-Idiotypic/biosynthesis , Antibodies, Anti-Idiotypic/immunology , Antibody Specificity , Antigens/biosynthesis , Antigens/chemistry , Antigens/immunology , Arthritis, Rheumatoid/immunology , Autoantibodies/immunology , Enzyme-Linked Immunosorbent Assay , Epitopes , Fibronectins/biosynthesis , Fibronectins/chemistry , Fibronectins/immunology , HEK293 Cells , Humans , Immunoglobulin G/blood , Immunoglobulin G/immunology , Molecular Mimicry , Molecular Sequence Data , Peptide Fragments/biosynthesis , Peptide Fragments/chemistry , Peptide Fragments/immunology , Peptide Library , Protein Binding , Protein Structure, Tertiary
9.
Anal Biochem ; 412(2): 210-6, 2011 May 15.
Article in English | MEDLINE | ID: mdl-21315061

ABSTRACT

Affinity reagents that are generated by phage display are typically subcloned into an expression vector for further biochemical characterization. This insert transfer process is time consuming and laborious especially if many inserts are to be subcloned. To simplify the transfer process, we have constructed a "drop-out" phagemid vector that can be rapidly converted to an expression vector by a simple restriction enzyme digestion with MfeI (to "drop-out" the gene III coding sequence), which generates alkaline phosphatase (AP) fusions of the affinity reagents on religation. Subsequently, restriction digestion with AscI drops out the AP coding region and religation generates affinity reagents with a C-terminal six-histidine tag. To validate the usefulness of this vector, four different human single chain Fragments of variable regions (scFv) were tested, three of which show specific binding to three zebrafish (Danio rerio) proteins, namely suppression of tumorigenicity 13, recoverin, and Ppib and the fourth binds to human Lactoferrin protein. For each of the constructs tested, the gene III and AP drop-out efficiency was between 90% and 100%. This vector is especially useful in speeding up the downstream screening of affinity reagents and bypassing the time-consuming subcloning experiments.


Subject(s)
Bacteriophages/genetics , Chromatography, Affinity , Gene Expression , Genetic Techniques , Genetic Vectors/genetics , Peptide Library , Alkaline Phosphatase , Animals , Blotting, Western , Electrophoresis, Polyacrylamide Gel , Humans , Indicators and Reagents , Recombinant Fusion Proteins/metabolism , Reproducibility of Results , Single-Chain Antibodies/isolation & purification , Zebrafish
10.
Mol Ther ; 18(8): 1471-81, 2010 Aug.
Article in English | MEDLINE | ID: mdl-20551911

ABSTRACT

Alzheimer's disease (AD) is a progressive dementing disorder characterized by age-related amyloid-beta (Abeta) deposition, neurofibrillary tangles, and synapse and neuronal loss. It is widely recognized that Abeta is a principal pathogenic mediator of AD. Our goal was to develop an immunotherapeutic approach, which would specifically lead to the clearance and/or neutralization of Abeta in the triple transgenic mouse model (3xTg-AD). These mice develop the amyloid and tangle pathologies and synaptic dysfunction reminiscent of human AD. Using a human single-chain variable fragment (scFv) antibody phage display library, a novel scFv antibody specific to Abeta was isolated, its activity characterized in vitro, and its open reading frame subsequently cloned into a recombinant adeno-associated virus (rAAV) vector. Three-month-old 3xTg-AD mice were intrahippocampally infused with serotype-1 rAAV vectors encoding Abeta-scFv or a control vector using convection-enhanced delivery (CED). Mice receiving rAAV1-Abeta-scFv harbored lower levels of insoluble Abeta and hyperphosphorylated tau, and exhibited improved cognitive function as measured by the Morris Water Maze (MWM) spatial memory task. These results underscore the potential of gene-based passive vaccination for AD, and provide further rationale for the development of Abeta-targeting strategies for this debilitating disease.


Subject(s)
Alzheimer Disease/pathology , Alzheimer Disease/therapy , Amyloid beta-Peptides/immunology , Dependovirus/genetics , Maze Learning/physiology , Single-Chain Antibodies/metabolism , Alzheimer Disease/metabolism , Animals , Cell Survival/genetics , Cell Survival/physiology , Cells, Cultured , Cricetinae , Enzyme-Linked Immunosorbent Assay , Humans , Immunohistochemistry , Male , Mice , Mice, Transgenic , Single-Chain Antibodies/genetics
11.
FEMS Immunol Med Microbiol ; 54(2): 195-202, 2008 Nov.
Article in English | MEDLINE | ID: mdl-18662314

ABSTRACT

The Candida albicans adhesin, Als3p, was identified as a potential cognate antigen for previously described human antibody fragments [single-chain variable fragment (scFv)] based on similarity of the binding pattern of the scFv to the distribution of this protein on the hyphal surface. Although all scFv bound avidly to wild type, scFv3 showed no detectable binding via immunofluorescence assay to strain 1843, containing a homozygous deletion of ALS3. Binding to the ALS3 reintegrant strain, 2322, was preserved, and scFv3 also bound to Saccharomyces cerevisiae expressing ALS3. Other scFv retained binding to 1843, but with a markedly altered pattern. To determine if scFv3 could interfere with Als3p function, adhesion assays were conducted using human epithelial or endothelial cells as target. Treatment of wild-type C. albicans with scFv3 reduced adhesion of the fungus to both cell types to levels comparable to the als3Delta/als3Delta mutant. These experiments confirm that phage display is a viable method to isolate human scFv specific to an antigen implicated in C. albicans virulence, and that the scFv interfere with adhesion to human cells. The altered pattern of immunostaining with other scFv that retain binding to the als3Delta/als3Delta mutant suggest that Als3p may also have a role in structural organization of the C. albicans cell surface.


Subject(s)
Candida albicans/immunology , Candida albicans/physiology , Fungal Proteins/immunology , Immunoglobulin Variable Region/immunology , Antibody Specificity , Antigens, Fungal/genetics , Antigens, Fungal/immunology , Antigens, Fungal/metabolism , Binding Sites, Antibody , Candida albicans/genetics , Cell Adhesion , Endothelial Cells/microbiology , Epithelial Cells/microbiology , Fluorescent Antibody Technique , Fungal Proteins/genetics , Fungal Proteins/metabolism , Humans , Immunoglobulin Variable Region/genetics , Recombinant Proteins/immunology
12.
J Proteome Res ; 7(5): 1994-2006, 2008 May.
Article in English | MEDLINE | ID: mdl-18361515

ABSTRACT

Saliva is a body fluid with important functions in oral and general health. A consortium of three research groups catalogued the proteins in human saliva collected as the ductal secretions: 1166 identifications--914 in parotid and 917 in submandibular/sublingual saliva--were made. The results showed that a high proportion of proteins that are found in plasma and/or tears are also present in saliva along with unique components. The proteins identified are involved in numerous molecular processes ranging from structural functions to enzymatic/catalytic activities. As expected, the majority mapped to the extracellular and secretory compartments. An immunoblot approach was used to validate the presence in saliva of a subset of the proteins identified by mass spectrometric approaches. These experiments focused on novel constituents and proteins for which the peptide evidence was relatively weak. Ultimately, information derived from the work reported here and related published studies can be used to translate blood-based clinical laboratory tests into a format that utilizes saliva. Additionally, a catalogue of the salivary proteome of healthy individuals allows future analyses of salivary samples from individuals with oral and systemic diseases, with the goal of identifying biomarkers with diagnostic and/or prognostic value for these conditions; another possibility is the discovery of therapeutic targets.


Subject(s)
Parotid Gland/chemistry , Proteome/analysis , Saliva/chemistry , Salivary Proteins and Peptides/analysis , Sublingual Gland/chemistry , Submandibular Gland/chemistry , Adult , Blood Proteins/analysis , Female , Humans , Male , Middle Aged , Molecular Sequence Data , Protein Array Analysis , Tears/chemistry
13.
Mol Ther ; 16(3): 481-6, 2008 Mar.
Article in English | MEDLINE | ID: mdl-18180775

ABSTRACT

A unifying characteristic of prion diseases is the conversion of a normal cellular protein (PrP(c)) to an abnormal pathogenic conformation, designated PrP(sc). Antibodies directed against PrP(c), when added to scrapie-infected cell cultures or passively administered in vivo, can result in elimination of PrP(sc) or prevent its replication, respectively. In our efforts to develop an approach with potential prophylactic utility we employed a recombinant adeno-associated vector type 2 (rAAV2) viral vector platform to express PrP(c)-specific single-chain fragment variable (scFv) antibodies within the central nervous system (CNS) of susceptible mice that were subsequently inoculated peripherally with infectious prions. Vector expressed scFvs delayed onset of prion pathogenesis as evidenced by improvements in clinical signs and rotarod performance, in extended incubation periods, and in decreased PrP(sc) burden in the CNS. This novel antibody delivery platform enables the in vivo translation of prion prophylactics to other species afflicted by transmissible spongiform encephalopathies (TSEs) and which also has relevance to the development of therapeutics for other protein-misfolding diseases such as Alzheimer's or Parkinson's disease.


Subject(s)
Antibodies/genetics , Central Nervous System/metabolism , Dependovirus/genetics , PrPC Proteins/immunology , Prion Diseases/prevention & control , Amino Acid Sequence , Animals , Antibodies/immunology , Blotting, Western , Cell Line , Densitometry , Endopeptidase K/metabolism , Enzyme-Linked Immunosorbent Assay , Genetic Vectors/genetics , Humans , Immunoblotting , Immunohistochemistry , Immunoprecipitation , Mice , Molecular Sequence Data , Prion Diseases/therapy , Sequence Homology, Amino Acid , Surface Plasmon Resonance , Transduction, Genetic
14.
Biochem Biophys Res Commun ; 349(4): 1198-205, 2006 Nov 03.
Article in English | MEDLINE | ID: mdl-16973126

ABSTRACT

Parkinson's disease (PD) is a common neurodegenerative disease of unknown etiology. Evidence suggests a role for protein misfolding in disease pathogenesis. One pathologic feature observed in dopaminergic neurons is the intracytoplasmic eosinophilic inclusions known as Lewy bodies. One component of Lewy bodies, the presynaptic protein, alpha-synuclein forms oligomers and higher order aggregates and is proposed to be involved in dopaminergic neuronal death. In an effort to discriminate between alpha-synuclein conformational forms as well as design potential disruptors of pathogenic misfolding we panned a human phage antibody library for anti-synuclein single chain antibodies (scFvs). We identified six scFvs which recognize different conformers of alpha-synuclein in both an ELISA and Western blot analysis. These scFvs may further our understanding of alpha-synuclein's role in PD.


Subject(s)
Immunoglobulin Variable Region/chemistry , Immunoglobulin Variable Region/immunology , alpha-Synuclein/chemistry , alpha-Synuclein/immunology , Humans , Immunoglobulin Variable Region/analysis , alpha-Synuclein/analysis
15.
J Struct Funct Genomics ; 6(2-3): 171-5, 2005.
Article in English | MEDLINE | ID: mdl-16211515

ABSTRACT

High throughput approaches to structural genomics requires expression, purification, and crystallization of proteins derived from predicted open reading frames cloned into a host organism, typically E. coli. Early results from this approach suggest that the success rate of obtaining well diffracting crystals from eukaryotic proteins is disappointingly low. A proven method of improving the odds of crystallization is formation of a complex with a conformation-stabilizing partner of known structure that is easily crystallized. Such complexes are also able to engage in different crystal contacts than the original protein by itself. Fab fragments derived from monoclonal antibodies have been successfully used for this purpose for a variety of proteins, however conventional methods for the isolation of monoclonal antibodies from hybridomas are time consuming and expensive. We are exploring the use of phage display to generate recombinant antibodies to target proteins that can be used to obtain co-complexes to facilitate crystallization and structural determination. We are using a large, human single-chain Fv (scFv) library to select for antibodies that bind to a panel of Leishmania major target proteins. Thirteen out of 16 target proteins yielded good binders after three rounds of enrichment. A total of 55 distinct scFvs were identified, with five targets each yielding at least five different scFvs. Individual clones were analyzed for binding specificity and soluble scFv can be readily produced and purified via the appended His(6) epitope tag. Using immunoaffinity chromatography, eight scFv target protein pairs were identified that exhibit stable complex formation and are suitable for co-crystallization trials.


Subject(s)
Immunoglobulin Fab Fragments/isolation & purification , Leishmania major/chemistry , Multiprotein Complexes/isolation & purification , Proteomics/methods , Protozoan Proteins/isolation & purification , Animals , Chromatography, Ion Exchange , Crystallization/methods , Electrophoresis, Polyacrylamide Gel , Enzyme-Linked Immunosorbent Assay , Gene Library , Humans , Immunoglobulin Fab Fragments/metabolism , Multiprotein Complexes/metabolism , Protozoan Proteins/metabolism
16.
Mol Biol Cell ; 16(9): 4202-13, 2005 Sep.
Article in English | MEDLINE | ID: mdl-15987734

ABSTRACT

RNA interference (RNAi) was used to characterize the requirement of protein glycosylation for cell membrane stability during cytokinesis in the early embryo. This screen targeted 13 enzymes or components of polypeptide sugar transferases that initiate either N-glycosylation or three different pathways of O-glycosylation. RNAi of genes in the mucin-type and epidermal growth factor-fringe glycosylation pathways did not affect cytokinesis. However, embryos deficient in N-glycosylation exhibited a variable inability to complete cytokinesis. The most potent block in early embryonic cell division was obtained by RNAi of the polypeptide xylose transferase (ppXyl-T), which is required to initiate the proteoglycan modification pathway. Two generations of ppXyl-T RNAi-feeding treatment reduced the body size, mobility, brood size, and life span of adult animals. Embryos escaping ppXyl-T and Gal-T2 RNAi lethality develop to adulthood but have cytokinesis-deficient offspring, suggesting that glycosyltransferases in the proteoglycan pathway are maternal proteins in the early embryo. Gal-T2::GFP fusions and anti-Gal-T2 antibodies revealed a perinuclear staining pattern, consistent with the localization of the Golgi apparatus. RNAi in green fluorescent protein (GFP)-tagged strains to follow tubulin, PIE-1, and chromatin showed that deficient proteoglycan biosynthesis uncouples the stability of newly formed cell membranes from cytokinesis, whereas cleavage furrow initiation, mitotic spindle function, karyokinesis, and partitioning of intrinsic components are intact.


Subject(s)
Caenorhabditis elegans Proteins/metabolism , Caenorhabditis elegans/embryology , Cytokinesis/physiology , Embryo, Nonmammalian/metabolism , Glycoproteins/metabolism , Glycosyltransferases/genetics , Amino Acid Sequence , Animals , Caenorhabditis elegans/metabolism , Cell Membrane , Embryo, Nonmammalian/cytology , Glycoproteins/biosynthesis , Glycosylation , Glycosyltransferases/metabolism , Mitosis/physiology , Molecular Sequence Data , Proteoglycans/metabolism , RNA Interference , Recombinant Proteins/genetics , Spindle Apparatus/physiology
17.
J Clin Microbiol ; 41(3): 1152-60, 2003 Mar.
Article in English | MEDLINE | ID: mdl-12624045

ABSTRACT

To identify antigens specific for the filamentous form of Candida albicans, a combinatorial phage display library expressing human immunoglobulin heavy and light chain variable regions was used to select phage clones capable of binding to the surfaces of viable C. albicans filaments. Eight distinct phage clones that bound specifically to filament surface antigens not expressed on blastoconidia were identified. Single-chain antibody variable fragments (scFv) derived from two of these phage clones (scFv5 and scFv12) were characterized in detail. Filament-specific antigen expression was detected by an indirect immunofluorescence assay. ScFv5 reacted with C. dubliniensis filaments, while scFv12 did not. Neither scFv reacted with C. glabrata, C. parapsilosis, C. rugosa, C. tropicalis, or Saccharomyces cerevisiae grown under conditions that stimulated filament formation in C. albicans and C. dubliniensis. Epitope detection by the two scFv was sensitive to proteinase K treatment but not to periodate treatment, indicating that the cognate epitopes were composed of protein. The antigens reactive with scFv5 and scFv12 were extractable from the cell surface with Zymolyase, but not with sodium dodecyl sulfate (SDS) and 2-mercaptoethanol, and migrated as polydisperse, high-molecular-weight bands on SDS-polyacrylamide gel electrophoresis gels. The epitopes were detected on clinical specimens obtained from infants with thrush and urinary candidiasis without passage of the organisms on laboratory media, confirming epitope expression in human infection. The availability of a monoclonal immunologic reagent that recognizes filaments from both C. albicans and C. dubliniensis and another specific only to C. albicans adds to the repertoire of potential diagnostic reagents for differentiation between these closely related species.


Subject(s)
Candida albicans/classification , Hyphae/immunology , Immunoglobulin Fragments , Antigens, Fungal/analysis , Blotting, Western , Candida albicans/immunology , Candida albicans/isolation & purification , Humans , Immunoglobulin Fragments/immunology , Peptide Fragments , Recombinant Proteins
18.
J Environ Manage ; 64(2): 153-69, 2002 Feb.
Article in English | MEDLINE | ID: mdl-11995238

ABSTRACT

We developed a forage allocation model using a deterministic, linear optimization module in a commercially available spreadsheet package to help resource managers in Theodore Roosevelt National Park (TRNP), North Dakota determine optimum numbers of four ungulate species, bison (Bison bison), elk (Cervus elaphus), mule deer (Odocoileus hemionus), and feral horses, in the Park. TRNP staff actively managed bison, elk, and feral horse numbers within bounds suggested by our model from 1983 to 1996. During this period, we measured vegetation at 8 grassland and 12 wooded sites at 1-3 year intervals to determine if model solutions were appropriate for maintaining stable conditions in important plant communities in the Park. The data we recorded at these sites indicated minimal change in plant communities from 1983 to 1996. Changes in most vegetation categories that we expected when animal numbers exceeded model optimums for short periods (decreases in coverage/stem numbers of palatable plant species, increases in bare ground or unpalatable plant species) did not occur consistently under high or low precipitation conditions. The lack of sensitivity of our model to decreases in overall production of palatable plant species that occurred due to drought, fire, expansion of black-tailed prairie dog (Cynomys ludovicianus) colonies, and the spread of leafy spurge (Euphorbia esula) in areas of the Park where we did not have monitoring sites suggested that the model under-estimated the total number of ungulates that the Park could support. Management for population levels of ungulates defined by the model probably led to over protection of common plant communities and insufficient protection of rare plant communities. Detecting changes in rare plant communities could have been accomplished by re-designing our vegetation monitoring program, but changing emphasis to protection of rare plants would have likely promoted under use of grazing-tolerant habitat types, dissatisfaction in tourists visiting the Park to see large mammals, and large increases in cost and intrusiveness of management activities such as fencing and control of ungulate populations. The model was a flawed representation of grazing dynamics in TRNP, but we believe it succeeded in making management personnel aware of the biological constraints they face when making management decisions.


Subject(s)
Bison , Conservation of Natural Resources , Deer , Horses , Models, Theoretical , Plants, Edible , Animals , Environmental Monitoring , Feeding Behavior , Female , Male , Population Dynamics
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