Your browser doesn't support javascript.
loading
Show: 20 | 50 | 100
Results 1 - 5 de 5
Filter
Add more filters










Database
Language
Publication year range
1.
ACS Chem Biol ; 15(2): 587-596, 2020 02 21.
Article in English | MEDLINE | ID: mdl-32003961

ABSTRACT

Capillary Morphogenesis Gene 2 protein (CMG2) is a transmembrane, integrin-like receptor and the primary receptor for the anthrax toxin. CMG2 also plays a role in angiogenic processes. However, the molecular mechanism that mediates the observed CMG2-related angiogenic effects is not fully elucidated. Previous studies have reported that CMG2 binds type IV collagen (Col-IV), a vital component of the vascular basement membrane, as well as other ECM proteins. Here, we further characterize the interaction between CMG2 and individual peptides from Col-IV and explore the effects of this interaction on angiogenesis. Using a peptide array, we observed that CMG2 preferentially binds peptide fragments of the NC1 (noncollagenous domain 1) domains of Col-IV. These domains are also known as the fragments arresten (from the α1 chain) and canstatin (from the α2 chain) and have documented antiangiogenic properties. A second peptide array was probed to map a putative peptide-binding epitope onto the Col-IV structure. A top hit from the initial array, a canstatin-derived peptide, binds to the CMG2 ligand-binding von Willebrand factor A (vWA) domain with a submicromolar affinity (peptide S16, Kd = 400 ± 200 nM). This peptide competes with anthrax protective antigen (PA) for CMG2 binding and does not bind CMG2 in the presence of EDTA. Together these data suggest that, like PA, S16 interacts with CMG2 at the metal-ion dependent adhesion site (MIDAS) of its vWA domain. CMG2 specifically mediates endocytic uptake of S16; both CMG2-/- endothelial cells and WT cells treated with PA show markedly reduced S16 uptake. Furthermore, S16 dramatically reduces directional endothelial cell migration with no impact on cell proliferation. These data demonstrate that this canstatin-derived peptide acts via CMG2 to elicit a marked effect on a critical process required for angiogenesis.


Subject(s)
Collagen Type IV/metabolism , Endocytosis/physiology , Peptide Fragments/metabolism , Receptors, Peptide/metabolism , Amino Acid Sequence , Animals , Binding Sites , Cell Line, Tumor , Cell Movement/drug effects , Endothelial Cells/metabolism , Humans , Mice , Protein Binding , Protein Domains , Receptors, Peptide/chemistry
2.
J Biol Chem ; 295(10): 2974-2983, 2020 03 06.
Article in English | MEDLINE | ID: mdl-31974166

ABSTRACT

Tau aggregation underlies neurodegeneration in Alzheimer's disease and related tauopathies. We and others have proposed that transcellular propagation of pathology is mediated by Tau prions, which are ordered protein assemblies that faithfully replicate in vivo and cause specific biological effects. The prion model predicts the release of aggregates from a first-order cell and subsequent uptake into a second-order cell. The assemblies then serve as templates for their own replication, a process termed "seeding." We have previously observed that heparan sulfate proteoglycans on the cell surface mediate the cellular uptake of Tau aggregates. This interaction is blocked by heparin, a sulfated glycosaminoglycan. Indeed, heparin-like molecules, or heparinoids, have previously been proposed as a treatment for PrP prion disorders. However, heparin is not ideal for managing chronic neurodegeneration, because it is difficult to synthesize in defined sizes, may have poor brain penetration because of its negative charge, and is a powerful anticoagulant. Therefore, we sought to generate an oligosaccharide that would bind Tau and block its cellular uptake and seeding, without exhibiting anticoagulation activity. We created a compound, SN7-13, from pentasaccharide units and tested it in a range of assays that measured direct binding of Tau to glycosaminoglycans and inhibition of Tau uptake and seeding in cells. SN7-13 does not inhibit coagulation, binds Tau with low nanomolar affinity, and inhibits cellular Tau aggregate propagation similarly to standard porcine heparin. This synthetic heparinoid could facilitate the development of agents to treat tauopathy.


Subject(s)
Heparin, Low-Molecular-Weight/metabolism , tau Proteins/metabolism , Animals , HEK293 Cells , Heparin, Low-Molecular-Weight/chemistry , Heparin, Low-Molecular-Weight/pharmacology , Hippocampus/metabolism , Humans , Mice , Neurons/metabolism , Partial Thromboplastin Time , Prion Diseases/metabolism , Prion Diseases/pathology , Protein Aggregates/drug effects , Protein Binding , Prothrombin Time , Recombinant Proteins/biosynthesis , Recombinant Proteins/chemistry , Recombinant Proteins/isolation & purification , tau Proteins/chemistry , tau Proteins/genetics
3.
Front Immunol ; 11: 561889, 2020.
Article in English | MEDLINE | ID: mdl-33542711

ABSTRACT

CD4+ T cells are crucial for effective repression and elimination of cancer cells. Despite a paucity of CD4+ T cell receptor (TCR) clinical studies, CD4+ T cells are primed to become important therapeutics as they help circumvent tumor antigen escape and guide multifactorial immune responses. However, because CD8+ T cells directly kill tumor cells, most research has focused on the attributes of CD8+ TCRs. Less is known about how TCR affinity and CD4 expression affect CD4+ T cell activation in full length TCR (flTCR) and TCR single chain signaling (TCR-SCS) formats. Here, we generated an affinity panel of TCRs from CD4+ T cells and expressed them in flTCR and three TCR-SCS formats modeled after chimeric antigen receptors (CARs) to understand the contributions of TCR-pMHCII affinity, TCR format, and coreceptor CD4 interactions on CD4+ T cell activation. Strikingly, the coreceptor CD4 inhibited intermediate and high affinity TCR-construct activation by Lck-dependent and -independent mechanisms. These inhibition mechanisms had unique affinity thresholds dependent on the TCR format. Intracellular construct formats affected the tetramer staining for each TCR as well as IL-2 production. IL-2 production was promoted by increased TCR-pMHCII affinity and the flTCR format. Thus, CD4+ T cell therapy development should consider TCR affinity, CD4 expression, and construct format.


Subject(s)
CD4 Antigens/metabolism , Lymphocyte Activation , Models, Immunological , Receptors, Antigen, T-Cell, alpha-beta/immunology , Receptors, Chimeric Antigen/immunology , Signal Transduction/immunology , T-Lymphocytes, Helper-Inducer/immunology , Animals , Antigens, Neoplasm/immunology , Histocompatibility Antigens Class II/immunology , Hybridomas , Immunotherapy, Adoptive/methods , Interleukin-2/metabolism , Lymphocyte Specific Protein Tyrosine Kinase p56(lck)/metabolism , Mice , Mice, Inbred C57BL , Mice, Transgenic , Receptors, Antigen, T-Cell, alpha-beta/genetics , Receptors, Chimeric Antigen/genetics , Transduction, Genetic , Yeasts/immunology
4.
J Med Chem ; 62(8): 3958-3970, 2019 04 25.
Article in English | MEDLINE | ID: mdl-30964669

ABSTRACT

We previously showed that a small molecule of natural origin, 1,2,3,4,6-penta- O-galloyl-ß-d-glucopyranose (PGG), binds to capillary morphogenesis gene 2 (CMG2) with a submicromolar IC50 and also has antiangiogenic activity in vitro and in vivo. In this work, we synthetized derivatives of PGG with different sugar cores and phenolic substituents and tested these as angiogenesis inhibitors. In a high-throughput Förster resonant energy transfer-based binding assay, we found that one of our synthetic analogues (1,2,3,4,6-penta- O-galloyl-ß-d-mannopyranose (PGM)), with mannose as central core and galloyl substituents, exhibit higher (up to 10×) affinity for CMG2 than the natural glucose prototype PGG and proved to be a potent angiogenesis inhibitor. These findings demonstrate that biochemical CMG2 binding in vitro predicts inhibition of endothelial cell migration ex vivo and antiangiogenic activity in vivo. The molecules herein described, and in particular PGM, might be useful prototypes for the development of novel agents for angiogenesis-dependent diseases, including blinding eye disease and cancer.


Subject(s)
Angiogenesis Inhibitors/chemistry , Hydrolyzable Tannins/chemistry , Receptors, Peptide/metabolism , Angiogenesis Inhibitors/metabolism , Angiogenesis Inhibitors/pharmacology , Animals , Hydrolyzable Tannins/metabolism , Hydrolyzable Tannins/pharmacology , Mannose/analogs & derivatives , Mannose/metabolism , Mannose/pharmacology , Mice , Mice, Inbred C57BL , Neovascularization, Physiologic/drug effects , Protein Binding , Receptors, Peptide/chemistry , Structure-Activity Relationship
5.
Anal Biochem ; 517: 23-30, 2017 Jan 15.
Article in English | MEDLINE | ID: mdl-27794422

ABSTRACT

Hepcidin is a small cysteine-rich signaling peptide that regulates blood serum iron concentrations [1-4]. Patients with chronic inflammation are known to have elevated levels of hepcidin in their blood and urine and often suffer from anemia as a result [5-10]. Measuring and quantifying the amount of active hepcidin in blood and urine can help to determine the cause and severity of the anemia thereby helping physicians determine the correct course of treatment [11-16]. We have developed a simple technique to isolate, chemically modify, and concentrate hepcidin from blood and urine coupled to high-pressure liquid chromatography mass spectrometry that can accurately and reproducibly measure and quantify the active hormone.


Subject(s)
Anemia/blood , Anemia/urine , Hepcidins/blood , Hepcidins/urine , Mass Spectrometry/methods , Chromatography, Liquid/methods , Female , Humans , Male
SELECTION OF CITATIONS
SEARCH DETAIL
...