Dragana Nesic | The Rockefeller University (original) (raw)

Papers by Dragana Nesic

Research paper thumbnail of Immunoprecipitation of CDT Complexes Containing Deletion Mutants of CdtA

<div><p>(A) The complexes (10 μg each) were immunoprecipitated with 10 μg anti-myc an... more <div><p>(A) The complexes (10 μg each) were immunoprecipitated with 10 μg anti-myc antibody (myc tag was fused to C-terminus of CdtC) in 150 mM NaCl, 20 mM HEPES (pH 7.5), 0.1% Nonidet P40 substitute, followed by exposure to protein G Sepharose beads, and subjected to nondenaturing SDS-PAGE.</p><p>(B) Nonspecific binding of CDT complexes to protein G Sepharose was not detected under the same experimental conditions. A, CdtA; B, CdtB; C, CdtC; A*, truncated CdtA; A1, holotoxin with CdtA Δ18−56; A2, holotoxin with CdtA Δ18−67; A3, holotoxin with CdtA Δ18−75; WT, wild-type; Ab, only anti-<i>myc</i> antibody, no CDT; IgG (H+L), nonreduced immunoglobulin G; IgG H, immunoglobulin G heavy chain; IgG L, immunoglobulin G light chain; SN, supernatant.</p></div

Research paper thumbnail of The Activity of CDT Holotoxin Labeled with Alexa Fluor 488

<div><p>(A) The toxicity of CDT holotoxin labeled with Alexa Fluor 488. HeLa cells we... more <div><p>(A) The toxicity of CDT holotoxin labeled with Alexa Fluor 488. HeLa cells were treated with 1 ng/ml (black) or 10 ng/ml (gray) concentration of unconjugated or Alexa Fluor 488−conjugated CDT for 3 hr at 37°C, 5% CO<sub>2</sub>. Cells were processed 48 hr after holotoxin treatment, and DNA content was measured by flow cytometry. The calculated percentages of cells in G0/G1, S, and G2/M are shown.</p><p>(B) Binding of CDT-Alexa Fluor 488 to cells. Harvested HeLa cells were exposed for 2 hr to 5 and 10 μg/ml concentration of wild-type or mutant CDT-Alexa Fluor 488. The histogram shows the binding of 5 or 10 μg/ml concentration of wild-type and mutant CDT-Alexa Fluor 488 conjugates to HeLa cells. Mock represents cells in buffer only (2% FCS in PBS), and control is goat anti-mouse IgG conjugated with Alexa Fluor 488, which does not bind to HeLa cells. The level of fluorescence was analyzed by flow cytometry. The relative levels of fluorescent labeling of wild-type and mutant CDT holotoxin was maintained to be nearly equivalent, with the mutant holotoxins (groove and aromatic patch) possessing a slightly higher level of labeling than the wild-type (Materials and Methods).</p></div

Research paper thumbnail of Structure of the Helicobacter pylori CagA Oncogene Bound to the Human Tumor Suppressor Apoptosis-stimulating Protein of p53-2

Research paper thumbnail of Structure of the integrin AlphaIIbBeta3-Abciximab complex

Research paper thumbnail of Overcoming the TCR Signalling Defect of β2-Microglobulin Deficient CD8+ T cells in Response to Wildtype Syngeneic MHC Class I

Immunoregulation in Health and Disease, 1997

Publisher Summary This chapter presents a study on the T-cell receptor (TCR) signaling defect of ... more Publisher Summary This chapter presents a study on the T-cell receptor (TCR) signaling defect of β2-microglobulin deficient CD8+ T cells. CD8+ T lymphocytes are stimulated by TCR mediated recognition of antigens presented as short peptides complexed to major histocompatibility complex (MHC) class I molecules. The study shows that the CD8+ T cells that are allowed to mature in the thymus of β2M–/– mice are tolerant to the syngeneic MHC class I, as demonstrated by the failure of β2M–/– mice to reject syngeneic β2M+/+ tumors in vivo. The partial response, consisting of cytotoxic attack in the absence of proliferation or cytokine secretion, can be converted to the full response by synergistic action of syngeneic MHC class I and phorbol esters. Phorbol ester-induced activation of the ras pathway seems to be responsible for reverting to the phenotype, suggesting that ras activation is required for cytokine secretion and proliferation of CD8+ cells. Protein kinase C (PKC) activation, on the other hand, could be selectively required for perforin/granzyme release. This conclusion is based on findings that PKC inhibitor affects cytolytic activity of β2M–/– CD8+ cells, with no effect on cytokine secretion. Thus, the induction of cytolysis and of cytokine secretion by effector CD8+ cells seems to be regulated by different TCR signaling pathways.

Research paper thumbnail of Electron microscopy shows that binding of monoclonal antibody PT25-2 primes integrin αIIbβ3 for ligand binding

Blood Advances, 2021

The murine monoclonal antibody (mAb) PT25-2 induces αIIbβ3 to bind ligand and initiate platelet a... more The murine monoclonal antibody (mAb) PT25-2 induces αIIbβ3 to bind ligand and initiate platelet aggregation. The underlying mechanism is unclear, because previous mutagenesis studies suggested that PT25-2 binds to the αIIb β propeller, a site distant from the Arg-Gly-Asp–binding pocket. To elucidate the mechanism, we studied the αIIbβ3–PT25-2 Fab complex by negative-stain and cryo-electron microscopy (EM). We found that PT25-2 binding results in αIIbβ3 partially exposing multiple ligand-induced binding site epitopes and adopting extended conformations without swing-out of the β3 hybrid domain. The cryo-EM structure showed PT25-2 binding to the αIIb residues identified by mutagenesis but also to 2 additional regions. Overlay of the cryo-EM structure with the bent αIIbβ3 crystal structure showed that binding of PT25-2 creates clashes with the αIIb calf-1/calf-2 domains, suggesting that PT25-2 selectively binds to partially or fully extended receptor conformations and prevents a return...

Research paper thumbnail of Mechanisms of Assembly and Cellular Interactions for the Bacterial Genotoxin CDT-2

Research paper thumbnail of African Trypanosomes Can Evade Immune Clearance by Sugar-Coating Antigenic Surfaces

Nature microbiology, 2018

Research paper thumbnail of Development of a Pure Small Molecule αVβ3 Antagonist

# Contributed equally αVβ3 has been implicated in the pathophysiology of important disorders, inc... more # Contributed equally αVβ3 has been implicated in the pathophysiology of important disorders, including osteoporosis, sickle cell disease, tumor angiogenesis, metastasis, virus invasion, glomerulonephritis, dermal and hepatic fibrosis, acute myelogenous leukemia, supravalvular aortic stenosis, bowel strictures, and T-cell lymphoma, but no αVβ3 antagonists have been approved for human therapy. The RGD-based αVβ3 antagonist MK-0429 (MK429) had favorable results in osteoporosis patients in Phase 2, but was not advanced, and the RGD-based antagonist cilengitide was studied in patients with malignancies, but did not demonstrate efficacy. Since RGD-based antagonists can under certain circumstances activate αVβ3, prime it to bind ligand, induce apoptosis, increase tumor angiogenesis and growth, and increase hepatic fibrosis, we sought to develop a pure αVβ3 antagonist that does not induce high affinity ligand binding or exposure of the epitope for mAb AP5, both of which depend on the swing-out of the β3 PSI domain. We built on Arnaout9s group9s studies of a variant fibronectin fragment with high affinity for αVβ3 (hFN10) that did not induce the β3 swing-out motion because a Trp substitution on the ligand formed a π-π interaction with β3 Y122 on the β1-α1 loop, thus preventing the latter9s movement toward the MIDAS, which is the trigger for β3 swing-out. To develop a small-molecule that mimics the effect of hFN10, we used inferences from a predicted docking pose of MK429 into the hFN10 binding site of the αVβ3 crystal structure to rationally design a compound with an aromatic ring that directly engages β3 Y122. Based on the results of molecular dynamics simulations, the aromatic group of this compound, TDI-4161, is only 4.4 A from Y122 (and forms a π-π interaction), which compares with 4.7 A for hFN10 and 7.0 A for MK429 (which does not form a π-π interaction). We analyzed compounds in 2 assays: 1. Adhesion of HEK293 cells expressing human αVβ3 (HEK-αVβ3) to fibrinogen (reported as the concentration producing 50% inhibition; IC50). 2. Exposure of the epitope for mAb AP5 (the concentration producing 50% exposure; EC50). The racemate of MK429 (rMK429) has an IC50=0.0036 µM and an EC50=0.019 µM, and cilengitide has an IC50=0.050 µM and an EC50=0.034 µM, indicating that both are potent αVβ3 antagonists and potent inducers of the αVβ3 active conformation. In sharp contrast, TDI-4161 has an IC50=0.022 µM and an EC50>10 µM, indicating that it does not induce the β3 conformational change even at 400X the IC50. TDI-4161 inhibited the binding of: purified αVβ3 to adenovirus 2 penton base (IC50 = 0.028 µM) and purified αVβ5 to vitronectin (IC50 =2.09 µM), but not purified αVβ6 to TGF-β1 Latency Associated Peptide even at 10 µM. TDI-4161inhibited adhesion of mouse endothelial cells containing murine αVβ3 to fibrinogen (IC50 = 0.437 µM). We tested TDI-41619s ability to 9prime" αVβ3 by: incubating it with HEK-αVβ3 at 10 µM, fixing the cells with paraformaldehyde, washing, incubating with fluorescent fibrinogen, and measuring bound fibrinogen by flow cytometry. The peptide RGDS (100 µM) increased fibrinogen binding from 8.7 ± 1.5 to 24.3 ± 1.4 GMFI (p Disclosures Filizola: Icahn School of Medicine at Mount Sinai: Patents & Royalties. Foley: Tri-Institutional Therapeutics Discovery Institute: Patents & Royalties.

Research paper thumbnail of Molecular Dynamicssimulations of A 2.8-Å Resolution Cryo-EM Structure of the αIIbβ3-Abciximab Complex

Research paper thumbnail of Cryo-Electron Microscopy Structure of the aIIbβ3-Abciximab Complex

Blood

DN, YZ, AS, & JL contributed equally, as did MF, TW, & BSC The αIIbβ3 antagonist antiplatelet dru... more DN, YZ, AS, & JL contributed equally, as did MF, TW, & BSC The αIIbβ3 antagonist antiplatelet drug abciximab, approved in 1994, is the chimeric antigen-binding fragment (Fab) comprising the variable regions of murine mAb 7E3 and human IgG1 and light chain κ constant domains. In studies involving thousands of patients undergoing percutaneous coronary interventions, abciximab decreased mortality and the risk of recurrent myocardial infarction. Mutagenesis studies conducted by us and others (Puzon-McLaughlin, JBC 2000; Takagi, Biochem 2002; Artoni, PNAS 2004) suggested that abciximab binds to the β3 C177-C184 specificity-determining loop (SDL) and Trp129 on the adjacent β1-α1 helix, and our negative-stain electron microscopy (EM) studies of the complex of mAb 7E3 with αIIbβ3 in nanodiscs (Choi, Blood 2013) supported its binding to the αIIbβ3 head domain. None of these studies, however, had the resolution to assess whether 7E3 or abciximab prevents fibrinogen binding by steric interfere...

Research paper thumbnail of The Interaction of Cross-Linked Fibrin Fragment D-Dimer with αIIbβ3 Requires Receptor Activation, Occurs at Sites Other Than the γ-404-411 Sequence, and Contributes to Clot Retraction

Blood

Background: The interaction between the fibrinogen (fbg) γ-chain sequence 404-411 and the 'RG... more Background: The interaction between the fibrinogen (fbg) γ-chain sequence 404-411 and the 'RGD pocket' formed by αIIb and β3 plays a major role in platelet (P) aggregation. It requires activation of αIIbβ3 when fbg is in solution, but not when fbg is immobilized. Less is known about the interaction of Ps with cross-linked fibrin, which presumably is the dominant form of fibrin(ogen) in human thrombi and the form that participates in clot retraction. αIIbβ3 is required for clot retraction since it is decreased or absent in Glanzmann thrombasthenia patients who lack a functional receptor. Paradoxically, clot retraction is essentially normal with fbg lacking γ-408-411 or in the presence EDTA, which eliminates fbg binding to the RGD pocket. We recently studied the interaction of αIIbβ3 with fbg fragments D100 (which has γ-404-411) and 'D98' (which essentially lacks γ-406-411) to identify fbg binding sites on αIIbβ3 other than the RGD pocket. We found that: 1. activated, ...

Research paper thumbnail of Novel Pure αVβ3 Integrin Antagonists That Do Not Induce Receptor Extension, Prime the Receptor, or Enhance Angiogenesis at Low Concentrations

The integrin αVβ3 receptor has been implicated in several important diseases, but no αVβ3 antagon... more The integrin αVβ3 receptor has been implicated in several important diseases, but no αVβ3 antagonists are approved for human therapy. One possible limitation of current small molecule antagonists is their ability to induce a major conformational change in the receptor that induces it to adopt a high-affinity ligand-binding state. In response, we used structural inferences from a pure peptide antagonist to design the small molecule pure antagonists TDI-4161 and TDI-3761. Both compounds inhibit αVβ3-mediated cell adhesion to αVβ3 ligands, but do not induce the conformational change as judged by antibody binding, electron microscopy, X-ray crystallography, and receptor priming studies. Both compounds demonstrated the favorable property of inhibiting bone resorption in vitro, supporting potential value in treating osteoporosis. Neither, however, had the unfavorable property of the αVβ3 antagonist cilengitide of enhancing aortic sprout angiogenesis at concentrations below its IC50s, a pr...

Research paper thumbnail of African trypanosomes evade immune clearance by O-glycosylation of the VSG surface coat

Nature microbiology, 2018

The African trypanosome Trypanosoma brucei spp. is a paradigm for antigenic variation, the orches... more The African trypanosome Trypanosoma brucei spp. is a paradigm for antigenic variation, the orchestrated alteration of cell surface molecules to evade host immunity. The parasite elicits robust antibody-mediated immune responses to its variant surface glycoprotein (VSG) coat, but evades immune clearance by repeatedly accessing a large genetic VSG repertoire and 'switching' to antigenically distinct VSGs. This persistent immune evasion has been ascribed exclusively to amino-acid variance on the VSG surface presented by a conserved underlying protein architecture. We establish here that this model does not account for the scope of VSG structural and biochemical diversity. The 1.4-Å-resolution crystal structure of the variant VSG3 manifests divergence in the tertiary fold and oligomeric state. The structure also reveals an O-linked carbohydrate on the top surface of VSG3. Mass spectrometric analysis indicates that this O-glycosylation site is heterogeneously occupied in VSG3 by ...

Research paper thumbnail of The differentiation and effector functions of CD8+ T cells /

Typescript. Thesis (Ph. D.)--New York University, Graduate School of Arts and Science, 1998. Incl... more Typescript. Thesis (Ph. D.)--New York University, Graduate School of Arts and Science, 1998. Includes bibliographical references (leaves : 132-167).

Research paper thumbnail of Nesic2014 SI

Research paper thumbnail of CagA of Helicobacter pylori interacts with and inhibits the Serine-Threonine Kinase PRK2

Cellular microbiology, Jan 3, 2015

CagA is a multifunctional toxin of Helicobacter pylori that is secreted into host epithelial cell... more CagA is a multifunctional toxin of Helicobacter pylori that is secreted into host epithelial cells by a type IV secretion system. Following host cell translocation CagA interferes with various host-cell signaling pathways. Most notably this toxin is involved in the disruption of apical-basolateral cell polarity and cell adhesion, as well as in the induction of cell proliferation, migration, and cell morphological changes. These are processes that also play an important role in epithelial-to-mesenchymal transition and cancer cell invasion. In fact, CagA is considered the only known bacterial oncoprotein. The cellular effects are triggered by a variety of CagA activities including the inhibition of serine-threonine kinase Par1b/MARK2 and the activation of tyrosine phosphatase SHP-2. Additionally, CagA was described to affect the activity of Src-family kinases and C-terminal src kinase (Csk) suggesting that interference with multiple cellular kinase- and phosphatase-associated signalin...

Research paper thumbnail of MHC class I is required for peripheral accumulation of CD8+ thymic emigrants

Journal of immunology (Baltimore, Md. : 1950), Jan 15, 1998

MHC molecules influence the fate of T lymphocytes at two important stages of their differentiatio... more MHC molecules influence the fate of T lymphocytes at two important stages of their differentiation. Recognition of self peptide/MHC complexes in the thymus determines whether immature T cells should live and mature into immunocompetent T cells or whether they should die. In the periphery, recognition of Ags presented by MHC molecules induces T cell activation, proliferation, and differentiation into effector/memory T cells. We describe in this work a third role that MHC molecules play in T cell physiology. CD8+ thymic emigrants require presence of MHC class I molecules in the periphery to seed the peripheral lymphoid organs. Numbers of CD8+ T cells are reduced severely in both the thymus and the periphery of beta2-microglobulin-deficient (beta2m[-/-]) mice. When grafted with wild-type (beta2m[+/+]) thymic epithelium, immature beta2m(-/-) T cells that populate the graft develop into functional mature CD8+ cells. However, significant numbers of peripheral CD8+ cells in grafted beta2m(...

Research paper thumbnail of The role of protein kinase C in CD8+ T lymphocyte effector responses

Journal of immunology (Baltimore, Md. : 1950), Jan 15, 1997

Rare CD8+ T cells present in beta2microglobulin-deficient (beta2m-/-) mice reject allogeneic tumo... more Rare CD8+ T cells present in beta2microglobulin-deficient (beta2m-/-) mice reject allogeneic tumors but not syngeneic wild-type tumors. The lack of syngeneic tumor rejection in vivo is correlated with a partial response of beta2m-/- CD8+ cell lines to syngeneic tumor cells in vitro. This partial response is characterized by perforin/granzyme-mediated cytolytic activity in the absence of cytokine secretion or proliferation. Allogeneic tumors induce cytolysis, cytokine secretion, and proliferation. Cytokine secretion may therefore be an important effector mechanism for tumor rejection by CD8+ T cells. To determine the missing signaling events needed for cytokine secretion as well as the events inducing the isolated cytotoxic response, we attempted to restore cytokine secretion of beta2m-/- CD8+ cells to syngeneic MHC class I. Phorbol ester and syngeneic tumor cells acted synergistically to induce full responsiveness of beta2m-/- CD8+ cells. However, this synergistic induction of cytok...

Research paper thumbnail of Factors influencing the patterns of T lymphocyte allorecognition1

Transplantation, 2002

Strong alloreactive T cell responses are a menace in transplantation surgery and their menagement... more Strong alloreactive T cell responses are a menace in transplantation surgery and their menagement requires understanding the basis of alloreactivity. Alloantigen recognition can be peptide independent, peptide specific, or peptide dependent. The mechanisms influencing each recognition pattern are largely unknown. Peptide dependence was examined in vitro by adding peptides to antigen processing-deficient cell line used as target in cytotoxic T cell assays. Responses to major histocompatibility complex (MHC) alleles most homologous to self were recently shown to be more peptide dependent than to those with lesser homology to self. Hence, peptide reactivity in vivo was estimated based on relative strengths of alloreactive responses to more homologous and less homologous MHC alleles. Alloreactive CD8+ TCR repertoire in beta2-microglobulin-deficient mice is preferentially peptide independent. The peptide-specific component is acquired as a function of wild-type thymic epithelium grafting. Irrespective of the presence of the peptide-specific component, in vivo alloantigenic priming was associated with a greater sensitivity to the MHC structure than was in vitro priming. Thymic positive selection and the mode of alloreactivity induction are the major independent factors determining the patterns of alloantigen recognition.

Research paper thumbnail of Immunoprecipitation of CDT Complexes Containing Deletion Mutants of CdtA

<div><p>(A) The complexes (10 μg each) were immunoprecipitated with 10 μg anti-myc an... more <div><p>(A) The complexes (10 μg each) were immunoprecipitated with 10 μg anti-myc antibody (myc tag was fused to C-terminus of CdtC) in 150 mM NaCl, 20 mM HEPES (pH 7.5), 0.1% Nonidet P40 substitute, followed by exposure to protein G Sepharose beads, and subjected to nondenaturing SDS-PAGE.</p><p>(B) Nonspecific binding of CDT complexes to protein G Sepharose was not detected under the same experimental conditions. A, CdtA; B, CdtB; C, CdtC; A*, truncated CdtA; A1, holotoxin with CdtA Δ18−56; A2, holotoxin with CdtA Δ18−67; A3, holotoxin with CdtA Δ18−75; WT, wild-type; Ab, only anti-<i>myc</i> antibody, no CDT; IgG (H+L), nonreduced immunoglobulin G; IgG H, immunoglobulin G heavy chain; IgG L, immunoglobulin G light chain; SN, supernatant.</p></div

Research paper thumbnail of The Activity of CDT Holotoxin Labeled with Alexa Fluor 488

<div><p>(A) The toxicity of CDT holotoxin labeled with Alexa Fluor 488. HeLa cells we... more <div><p>(A) The toxicity of CDT holotoxin labeled with Alexa Fluor 488. HeLa cells were treated with 1 ng/ml (black) or 10 ng/ml (gray) concentration of unconjugated or Alexa Fluor 488−conjugated CDT for 3 hr at 37°C, 5% CO<sub>2</sub>. Cells were processed 48 hr after holotoxin treatment, and DNA content was measured by flow cytometry. The calculated percentages of cells in G0/G1, S, and G2/M are shown.</p><p>(B) Binding of CDT-Alexa Fluor 488 to cells. Harvested HeLa cells were exposed for 2 hr to 5 and 10 μg/ml concentration of wild-type or mutant CDT-Alexa Fluor 488. The histogram shows the binding of 5 or 10 μg/ml concentration of wild-type and mutant CDT-Alexa Fluor 488 conjugates to HeLa cells. Mock represents cells in buffer only (2% FCS in PBS), and control is goat anti-mouse IgG conjugated with Alexa Fluor 488, which does not bind to HeLa cells. The level of fluorescence was analyzed by flow cytometry. The relative levels of fluorescent labeling of wild-type and mutant CDT holotoxin was maintained to be nearly equivalent, with the mutant holotoxins (groove and aromatic patch) possessing a slightly higher level of labeling than the wild-type (Materials and Methods).</p></div

Research paper thumbnail of Structure of the Helicobacter pylori CagA Oncogene Bound to the Human Tumor Suppressor Apoptosis-stimulating Protein of p53-2

Research paper thumbnail of Structure of the integrin AlphaIIbBeta3-Abciximab complex

Research paper thumbnail of Overcoming the TCR Signalling Defect of β2-Microglobulin Deficient CD8+ T cells in Response to Wildtype Syngeneic MHC Class I

Immunoregulation in Health and Disease, 1997

Publisher Summary This chapter presents a study on the T-cell receptor (TCR) signaling defect of ... more Publisher Summary This chapter presents a study on the T-cell receptor (TCR) signaling defect of β2-microglobulin deficient CD8+ T cells. CD8+ T lymphocytes are stimulated by TCR mediated recognition of antigens presented as short peptides complexed to major histocompatibility complex (MHC) class I molecules. The study shows that the CD8+ T cells that are allowed to mature in the thymus of β2M–/– mice are tolerant to the syngeneic MHC class I, as demonstrated by the failure of β2M–/– mice to reject syngeneic β2M+/+ tumors in vivo. The partial response, consisting of cytotoxic attack in the absence of proliferation or cytokine secretion, can be converted to the full response by synergistic action of syngeneic MHC class I and phorbol esters. Phorbol ester-induced activation of the ras pathway seems to be responsible for reverting to the phenotype, suggesting that ras activation is required for cytokine secretion and proliferation of CD8+ cells. Protein kinase C (PKC) activation, on the other hand, could be selectively required for perforin/granzyme release. This conclusion is based on findings that PKC inhibitor affects cytolytic activity of β2M–/– CD8+ cells, with no effect on cytokine secretion. Thus, the induction of cytolysis and of cytokine secretion by effector CD8+ cells seems to be regulated by different TCR signaling pathways.

Research paper thumbnail of Electron microscopy shows that binding of monoclonal antibody PT25-2 primes integrin αIIbβ3 for ligand binding

Blood Advances, 2021

The murine monoclonal antibody (mAb) PT25-2 induces αIIbβ3 to bind ligand and initiate platelet a... more The murine monoclonal antibody (mAb) PT25-2 induces αIIbβ3 to bind ligand and initiate platelet aggregation. The underlying mechanism is unclear, because previous mutagenesis studies suggested that PT25-2 binds to the αIIb β propeller, a site distant from the Arg-Gly-Asp–binding pocket. To elucidate the mechanism, we studied the αIIbβ3–PT25-2 Fab complex by negative-stain and cryo-electron microscopy (EM). We found that PT25-2 binding results in αIIbβ3 partially exposing multiple ligand-induced binding site epitopes and adopting extended conformations without swing-out of the β3 hybrid domain. The cryo-EM structure showed PT25-2 binding to the αIIb residues identified by mutagenesis but also to 2 additional regions. Overlay of the cryo-EM structure with the bent αIIbβ3 crystal structure showed that binding of PT25-2 creates clashes with the αIIb calf-1/calf-2 domains, suggesting that PT25-2 selectively binds to partially or fully extended receptor conformations and prevents a return...

Research paper thumbnail of Mechanisms of Assembly and Cellular Interactions for the Bacterial Genotoxin CDT-2

Research paper thumbnail of African Trypanosomes Can Evade Immune Clearance by Sugar-Coating Antigenic Surfaces

Nature microbiology, 2018

Research paper thumbnail of Development of a Pure Small Molecule αVβ3 Antagonist

# Contributed equally αVβ3 has been implicated in the pathophysiology of important disorders, inc... more # Contributed equally αVβ3 has been implicated in the pathophysiology of important disorders, including osteoporosis, sickle cell disease, tumor angiogenesis, metastasis, virus invasion, glomerulonephritis, dermal and hepatic fibrosis, acute myelogenous leukemia, supravalvular aortic stenosis, bowel strictures, and T-cell lymphoma, but no αVβ3 antagonists have been approved for human therapy. The RGD-based αVβ3 antagonist MK-0429 (MK429) had favorable results in osteoporosis patients in Phase 2, but was not advanced, and the RGD-based antagonist cilengitide was studied in patients with malignancies, but did not demonstrate efficacy. Since RGD-based antagonists can under certain circumstances activate αVβ3, prime it to bind ligand, induce apoptosis, increase tumor angiogenesis and growth, and increase hepatic fibrosis, we sought to develop a pure αVβ3 antagonist that does not induce high affinity ligand binding or exposure of the epitope for mAb AP5, both of which depend on the swing-out of the β3 PSI domain. We built on Arnaout9s group9s studies of a variant fibronectin fragment with high affinity for αVβ3 (hFN10) that did not induce the β3 swing-out motion because a Trp substitution on the ligand formed a π-π interaction with β3 Y122 on the β1-α1 loop, thus preventing the latter9s movement toward the MIDAS, which is the trigger for β3 swing-out. To develop a small-molecule that mimics the effect of hFN10, we used inferences from a predicted docking pose of MK429 into the hFN10 binding site of the αVβ3 crystal structure to rationally design a compound with an aromatic ring that directly engages β3 Y122. Based on the results of molecular dynamics simulations, the aromatic group of this compound, TDI-4161, is only 4.4 A from Y122 (and forms a π-π interaction), which compares with 4.7 A for hFN10 and 7.0 A for MK429 (which does not form a π-π interaction). We analyzed compounds in 2 assays: 1. Adhesion of HEK293 cells expressing human αVβ3 (HEK-αVβ3) to fibrinogen (reported as the concentration producing 50% inhibition; IC50). 2. Exposure of the epitope for mAb AP5 (the concentration producing 50% exposure; EC50). The racemate of MK429 (rMK429) has an IC50=0.0036 µM and an EC50=0.019 µM, and cilengitide has an IC50=0.050 µM and an EC50=0.034 µM, indicating that both are potent αVβ3 antagonists and potent inducers of the αVβ3 active conformation. In sharp contrast, TDI-4161 has an IC50=0.022 µM and an EC50>10 µM, indicating that it does not induce the β3 conformational change even at 400X the IC50. TDI-4161 inhibited the binding of: purified αVβ3 to adenovirus 2 penton base (IC50 = 0.028 µM) and purified αVβ5 to vitronectin (IC50 =2.09 µM), but not purified αVβ6 to TGF-β1 Latency Associated Peptide even at 10 µM. TDI-4161inhibited adhesion of mouse endothelial cells containing murine αVβ3 to fibrinogen (IC50 = 0.437 µM). We tested TDI-41619s ability to 9prime" αVβ3 by: incubating it with HEK-αVβ3 at 10 µM, fixing the cells with paraformaldehyde, washing, incubating with fluorescent fibrinogen, and measuring bound fibrinogen by flow cytometry. The peptide RGDS (100 µM) increased fibrinogen binding from 8.7 ± 1.5 to 24.3 ± 1.4 GMFI (p Disclosures Filizola: Icahn School of Medicine at Mount Sinai: Patents & Royalties. Foley: Tri-Institutional Therapeutics Discovery Institute: Patents & Royalties.

Research paper thumbnail of Molecular Dynamicssimulations of A 2.8-Å Resolution Cryo-EM Structure of the αIIbβ3-Abciximab Complex

Research paper thumbnail of Cryo-Electron Microscopy Structure of the aIIbβ3-Abciximab Complex

Blood

DN, YZ, AS, & JL contributed equally, as did MF, TW, & BSC The αIIbβ3 antagonist antiplatelet dru... more DN, YZ, AS, & JL contributed equally, as did MF, TW, & BSC The αIIbβ3 antagonist antiplatelet drug abciximab, approved in 1994, is the chimeric antigen-binding fragment (Fab) comprising the variable regions of murine mAb 7E3 and human IgG1 and light chain κ constant domains. In studies involving thousands of patients undergoing percutaneous coronary interventions, abciximab decreased mortality and the risk of recurrent myocardial infarction. Mutagenesis studies conducted by us and others (Puzon-McLaughlin, JBC 2000; Takagi, Biochem 2002; Artoni, PNAS 2004) suggested that abciximab binds to the β3 C177-C184 specificity-determining loop (SDL) and Trp129 on the adjacent β1-α1 helix, and our negative-stain electron microscopy (EM) studies of the complex of mAb 7E3 with αIIbβ3 in nanodiscs (Choi, Blood 2013) supported its binding to the αIIbβ3 head domain. None of these studies, however, had the resolution to assess whether 7E3 or abciximab prevents fibrinogen binding by steric interfere...

Research paper thumbnail of The Interaction of Cross-Linked Fibrin Fragment D-Dimer with αIIbβ3 Requires Receptor Activation, Occurs at Sites Other Than the γ-404-411 Sequence, and Contributes to Clot Retraction

Blood

Background: The interaction between the fibrinogen (fbg) γ-chain sequence 404-411 and the 'RG... more Background: The interaction between the fibrinogen (fbg) γ-chain sequence 404-411 and the 'RGD pocket' formed by αIIb and β3 plays a major role in platelet (P) aggregation. It requires activation of αIIbβ3 when fbg is in solution, but not when fbg is immobilized. Less is known about the interaction of Ps with cross-linked fibrin, which presumably is the dominant form of fibrin(ogen) in human thrombi and the form that participates in clot retraction. αIIbβ3 is required for clot retraction since it is decreased or absent in Glanzmann thrombasthenia patients who lack a functional receptor. Paradoxically, clot retraction is essentially normal with fbg lacking γ-408-411 or in the presence EDTA, which eliminates fbg binding to the RGD pocket. We recently studied the interaction of αIIbβ3 with fbg fragments D100 (which has γ-404-411) and 'D98' (which essentially lacks γ-406-411) to identify fbg binding sites on αIIbβ3 other than the RGD pocket. We found that: 1. activated, ...

Research paper thumbnail of Novel Pure αVβ3 Integrin Antagonists That Do Not Induce Receptor Extension, Prime the Receptor, or Enhance Angiogenesis at Low Concentrations

The integrin αVβ3 receptor has been implicated in several important diseases, but no αVβ3 antagon... more The integrin αVβ3 receptor has been implicated in several important diseases, but no αVβ3 antagonists are approved for human therapy. One possible limitation of current small molecule antagonists is their ability to induce a major conformational change in the receptor that induces it to adopt a high-affinity ligand-binding state. In response, we used structural inferences from a pure peptide antagonist to design the small molecule pure antagonists TDI-4161 and TDI-3761. Both compounds inhibit αVβ3-mediated cell adhesion to αVβ3 ligands, but do not induce the conformational change as judged by antibody binding, electron microscopy, X-ray crystallography, and receptor priming studies. Both compounds demonstrated the favorable property of inhibiting bone resorption in vitro, supporting potential value in treating osteoporosis. Neither, however, had the unfavorable property of the αVβ3 antagonist cilengitide of enhancing aortic sprout angiogenesis at concentrations below its IC50s, a pr...

Research paper thumbnail of African trypanosomes evade immune clearance by O-glycosylation of the VSG surface coat

Nature microbiology, 2018

The African trypanosome Trypanosoma brucei spp. is a paradigm for antigenic variation, the orches... more The African trypanosome Trypanosoma brucei spp. is a paradigm for antigenic variation, the orchestrated alteration of cell surface molecules to evade host immunity. The parasite elicits robust antibody-mediated immune responses to its variant surface glycoprotein (VSG) coat, but evades immune clearance by repeatedly accessing a large genetic VSG repertoire and 'switching' to antigenically distinct VSGs. This persistent immune evasion has been ascribed exclusively to amino-acid variance on the VSG surface presented by a conserved underlying protein architecture. We establish here that this model does not account for the scope of VSG structural and biochemical diversity. The 1.4-Å-resolution crystal structure of the variant VSG3 manifests divergence in the tertiary fold and oligomeric state. The structure also reveals an O-linked carbohydrate on the top surface of VSG3. Mass spectrometric analysis indicates that this O-glycosylation site is heterogeneously occupied in VSG3 by ...

Research paper thumbnail of The differentiation and effector functions of CD8+ T cells /

Typescript. Thesis (Ph. D.)--New York University, Graduate School of Arts and Science, 1998. Incl... more Typescript. Thesis (Ph. D.)--New York University, Graduate School of Arts and Science, 1998. Includes bibliographical references (leaves : 132-167).

Research paper thumbnail of Nesic2014 SI

Research paper thumbnail of CagA of Helicobacter pylori interacts with and inhibits the Serine-Threonine Kinase PRK2

Cellular microbiology, Jan 3, 2015

CagA is a multifunctional toxin of Helicobacter pylori that is secreted into host epithelial cell... more CagA is a multifunctional toxin of Helicobacter pylori that is secreted into host epithelial cells by a type IV secretion system. Following host cell translocation CagA interferes with various host-cell signaling pathways. Most notably this toxin is involved in the disruption of apical-basolateral cell polarity and cell adhesion, as well as in the induction of cell proliferation, migration, and cell morphological changes. These are processes that also play an important role in epithelial-to-mesenchymal transition and cancer cell invasion. In fact, CagA is considered the only known bacterial oncoprotein. The cellular effects are triggered by a variety of CagA activities including the inhibition of serine-threonine kinase Par1b/MARK2 and the activation of tyrosine phosphatase SHP-2. Additionally, CagA was described to affect the activity of Src-family kinases and C-terminal src kinase (Csk) suggesting that interference with multiple cellular kinase- and phosphatase-associated signalin...

Research paper thumbnail of MHC class I is required for peripheral accumulation of CD8+ thymic emigrants

Journal of immunology (Baltimore, Md. : 1950), Jan 15, 1998

MHC molecules influence the fate of T lymphocytes at two important stages of their differentiatio... more MHC molecules influence the fate of T lymphocytes at two important stages of their differentiation. Recognition of self peptide/MHC complexes in the thymus determines whether immature T cells should live and mature into immunocompetent T cells or whether they should die. In the periphery, recognition of Ags presented by MHC molecules induces T cell activation, proliferation, and differentiation into effector/memory T cells. We describe in this work a third role that MHC molecules play in T cell physiology. CD8+ thymic emigrants require presence of MHC class I molecules in the periphery to seed the peripheral lymphoid organs. Numbers of CD8+ T cells are reduced severely in both the thymus and the periphery of beta2-microglobulin-deficient (beta2m[-/-]) mice. When grafted with wild-type (beta2m[+/+]) thymic epithelium, immature beta2m(-/-) T cells that populate the graft develop into functional mature CD8+ cells. However, significant numbers of peripheral CD8+ cells in grafted beta2m(...

Research paper thumbnail of The role of protein kinase C in CD8+ T lymphocyte effector responses

Journal of immunology (Baltimore, Md. : 1950), Jan 15, 1997

Rare CD8+ T cells present in beta2microglobulin-deficient (beta2m-/-) mice reject allogeneic tumo... more Rare CD8+ T cells present in beta2microglobulin-deficient (beta2m-/-) mice reject allogeneic tumors but not syngeneic wild-type tumors. The lack of syngeneic tumor rejection in vivo is correlated with a partial response of beta2m-/- CD8+ cell lines to syngeneic tumor cells in vitro. This partial response is characterized by perforin/granzyme-mediated cytolytic activity in the absence of cytokine secretion or proliferation. Allogeneic tumors induce cytolysis, cytokine secretion, and proliferation. Cytokine secretion may therefore be an important effector mechanism for tumor rejection by CD8+ T cells. To determine the missing signaling events needed for cytokine secretion as well as the events inducing the isolated cytotoxic response, we attempted to restore cytokine secretion of beta2m-/- CD8+ cells to syngeneic MHC class I. Phorbol ester and syngeneic tumor cells acted synergistically to induce full responsiveness of beta2m-/- CD8+ cells. However, this synergistic induction of cytok...

Research paper thumbnail of Factors influencing the patterns of T lymphocyte allorecognition1

Transplantation, 2002

Strong alloreactive T cell responses are a menace in transplantation surgery and their menagement... more Strong alloreactive T cell responses are a menace in transplantation surgery and their menagement requires understanding the basis of alloreactivity. Alloantigen recognition can be peptide independent, peptide specific, or peptide dependent. The mechanisms influencing each recognition pattern are largely unknown. Peptide dependence was examined in vitro by adding peptides to antigen processing-deficient cell line used as target in cytotoxic T cell assays. Responses to major histocompatibility complex (MHC) alleles most homologous to self were recently shown to be more peptide dependent than to those with lesser homology to self. Hence, peptide reactivity in vivo was estimated based on relative strengths of alloreactive responses to more homologous and less homologous MHC alleles. Alloreactive CD8+ TCR repertoire in beta2-microglobulin-deficient mice is preferentially peptide independent. The peptide-specific component is acquired as a function of wild-type thymic epithelium grafting. Irrespective of the presence of the peptide-specific component, in vivo alloantigenic priming was associated with a greater sensitivity to the MHC structure than was in vitro priming. Thymic positive selection and the mode of alloreactivity induction are the major independent factors determining the patterns of alloantigen recognition.