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1.
Oncotarget ; 7(33): 53414-53429, 2016 Aug 16.
Article in English | MEDLINE | ID: mdl-27462785

ABSTRACT

Unique molecular properties of species D adenoviruses (Ads)-the most diverse yet underexplored group of Ads-have been used to develop improved gene vectors. The low seroprevalence in humans of adenovirus serotype 43 (Ad43), an otherwise unstudied species D Ad, identified this rare serotype as an attractive new human gene therapy vector platform. Thus, in this study we wished to assess biological properties of Ad43 essential to its vectorization. We found that (1) Ad43 virions do not bind blood coagulation factor X and cause low random transduction upon vascular delivery; (2) they clear host tissues more quickly than do traditionally used Ad5 vectors; (3) Ad43 uses CD46 as primary receptor; (4) Ad43 can use integrins as alternative primary receptors. As the first step toward vectorization of Ad43, we demonstrated that the primary receptor specificity of the Ad43 fiber can be altered to achieve infection via Her2, an established oncotarget. Whereas this modification required use of the Ad5 fiber shaft, the presence of this domain in chimeric virions did not make them susceptible for neutralization by anti-Ad5 antibodies.


Subject(s)
Adenoviridae/genetics , Genetic Therapy/methods , Genetic Vectors , Animals , Humans , Mice
2.
Cancer Cell ; 28(5): 610-622, 2015 Nov 09.
Article in English | MEDLINE | ID: mdl-26481148

ABSTRACT

While recombinant human erythropoietin (rhEpo) has been widely used to treat anemia in cancer patients, concerns about its adverse effects on patient survival have emerged. A lack of correlation between expression of the canonical EpoR and rhEpo's effects on cancer cells prompted us to consider the existence of an alternative Epo receptor. Here, we identified EphB4 as an Epo receptor that triggers downstream signaling via STAT3 and promotes rhEpo-induced tumor growth and progression. In human ovarian and breast cancer samples, expression of EphB4 rather than the canonical EpoR correlated with decreased disease-specific survival in rhEpo-treated patients. These results identify EphB4 as a critical mediator of erythropoietin-induced tumor progression and further provide clinically significant dimension to the biology of erythropoietin.


Subject(s)
Breast Neoplasms/genetics , Erythropoietin/pharmacology , Gene Expression Regulation, Neoplastic/drug effects , Ovarian Neoplasms/genetics , Receptor, EphB4/genetics , Adult , Aged , Aged, 80 and over , Animals , Blotting, Western , Breast Neoplasms/metabolism , Breast Neoplasms/pathology , Cell Line, Tumor , Disease Progression , Erythropoietin/genetics , Female , Humans , Kaplan-Meier Estimate , MCF-7 Cells , Mice, Inbred C57BL , Mice, Nude , Middle Aged , Ovarian Neoplasms/metabolism , Ovarian Neoplasms/pathology , Protein Binding/drug effects , Receptor, EphB4/metabolism , Receptors, Erythropoietin/genetics , Receptors, Erythropoietin/metabolism , Recombinant Proteins/pharmacology , Reverse Transcriptase Polymerase Chain Reaction , STAT3 Transcription Factor/genetics , STAT3 Transcription Factor/metabolism , Young Adult
3.
Nat Struct Mol Biol ; 21(2): 180-8, 2014 Feb.
Article in English | MEDLINE | ID: mdl-24440983

ABSTRACT

FGFR2-expressing human cancer cells with low concentrations of the adaptor protein Grb2 show high prevalence for metastatic outcome. In nonstimulated cells, the SH3 domain (and not the SH2 domains) of Plcγ1 directly competes for a binding site at the very C terminus of FGFR2 with the C-terminal SH3 domain of Grb2. Reduction of Grb2 concentration permits Plcγ1 access to the receptor. Recruitment of Plcγ1 in this way is sufficient to upregulate phospholipase activity. This results in elevated phosphatidylinositol 4,5-bisphosphate turnover and intracellular calcium levels, thus leading to increased cell motility and promotion of cell-invasive behavior in the absence of extracellular receptor stimulation. Therefore, metastatic outcome can be dictated by the constitutive competition between Grb2 and Plcγ1 for the phosphorylation-independent binding site on FGFR2.


Subject(s)
GRB2 Adaptor Protein/physiology , Phospholipase C gamma/physiology , Phospholipases/physiology , Receptor, Fibroblast Growth Factor, Type 2/metabolism , Binding Sites , Binding, Competitive , Cell Line, Tumor , GRB2 Adaptor Protein/metabolism , HEK293 Cells , Humans , Models, Genetic , Neoplasm Invasiveness/genetics , Phospholipase C gamma/metabolism , Protein Structure, Tertiary
4.
Cell ; 154(3): 556-68, 2013 Aug 01.
Article in English | MEDLINE | ID: mdl-23911321

ABSTRACT

Skp2 E3 ligase is overexpressed in numerous human cancers and plays a critical role in cell-cycle progression, senescence, metabolism, cancer progression, and metastasis. In the present study, we identified a specific Skp2 inhibitor using high-throughput in silico screening of large and diverse chemical libraries. This Skp2 inhibitor selectively suppresses Skp2 E3 ligase activity, but not activity of other SCF complexes. It also phenocopies the effects observed upon genetic Skp2 deficiency, such as suppressing survival and Akt-mediated glycolysis and triggering p53-independent cellular senescence. Strikingly, we discovered a critical function of Skp2 in positively regulating cancer stem cell populations and self-renewal ability through genetic and pharmacological approaches. Notably, Skp2 inhibitor exhibits potent antitumor activities in multiple animal models and cooperates with chemotherapeutic agents to reduce cancer cell survival. Our study thus provides pharmacological evidence that Skp2 is a promising target for restricting cancer stem cell and cancer progression.


Subject(s)
Antineoplastic Agents/pharmacology , Drug Discovery , Neoplasms/enzymology , Neoplastic Stem Cells/drug effects , S-Phase Kinase-Associated Proteins/antagonists & inhibitors , Ubiquitin-Protein Ligases/antagonists & inhibitors , Animals , Antineoplastic Agents/chemistry , Disease Models, Animal , Drug Screening Assays, Antitumor , Genes, p53 , Glycolysis/drug effects , Humans , Mice , Mice, Nude , Models, Molecular , Multienzyme Complexes/antagonists & inhibitors , Multienzyme Complexes/chemistry , Multienzyme Complexes/metabolism , Neoplasm Transplantation , Neoplasms/drug therapy , Neoplasms/genetics , Neoplastic Stem Cells/metabolism , S-Phase Kinase-Associated Proteins/chemistry , S-Phase Kinase-Associated Proteins/metabolism , Small Molecule Libraries , Structure-Activity Relationship , Transplantation, Heterologous , Ubiquitin-Protein Ligases/chemistry , Ubiquitin-Protein Ligases/metabolism
5.
Int J Biol Macromol ; 57: 69-75, 2013 Jun.
Article in English | MEDLINE | ID: mdl-23470436

ABSTRACT

The binding of the well-studied DNA aptamer aHt (5'-ATACCAGTCTATTCAATTGGGCCCGTCCGTAT GGTGGGTGTGCTGGCCAG-3'), which has been demonstrated to recognize human vascular endothelial growth factor (VEGF165) to recombinant VEGF was characterized using fluorescence anisotropy, isothermal titration calorimetry and analytical ultracentrifugation. The negatively-charged DNA aptamer is selective for VEGF and does not recognize positively-charged hen egg lysozyme, or bovine serum albumin. In contrast to the VEGF association of the previously-described aV DNA aptamer, where the binding is enthalpically driven and sequence-specific, the binding of the aHt aptamer to VEGF is entropically-driven and not abolished by scrambling of the sequence.


Subject(s)
Aptamers, Nucleotide/chemistry , Vascular Endothelial Growth Factor A/chemistry , Animals , Aptamers, Nucleotide/chemical synthesis , Cattle , Chickens , Humans , Muramidase/chemistry , Serum Albumin, Bovine/chemistry , Vascular Endothelial Growth Factor A/antagonists & inhibitors
6.
PLoS One ; 8(2): e57859, 2013.
Article in English | MEDLINE | ID: mdl-23469090

ABSTRACT

The polyunsaturated fatty acid (PUFA) synthases from deep-sea bacteria invariably contain multiple acyl carrier protein (ACP) domains in tandem. This conserved tandem arrangement has been implicated in both amplification of fatty acid production (additive effect) and in structural stabilization of the multidomain protein (synergistic effect). While the more accepted model is one in which domains act independently, recent reports suggest that ACP domains may form higher oligomers. Elucidating the three-dimensional structure of tandem arrangements may therefore give important insights into the functional relevance of these structures, and hence guide bioengineering strategies. In an effort to elucidate the three-dimensional structure of tandem repeats from deep-sea anaerobic bacteria, we have expressed and purified a fragment consisting of five tandem ACP domains from the PUFA synthase from Photobacterium profundum. Analysis of the tandem ACP fragment by analytical gel filtration chromatography showed a retention time suggestive of a multimeric protein. However, small angle X-ray scattering (SAXS) revealed that the multi-ACP fragment is an elongated monomer which does not form a globular unit. Stokes radii calculated from atomic monomeric SAXS models were comparable to those measured by analytical gel filtration chromatography, showing that in the gel filtration experiment, the molecular weight was overestimated due to the elongated protein shape. Thermal denaturation monitored by circular dichroism showed that unfolding of the tandem construct was not cooperative, and that the tandem arrangement did not stabilize the protein. Taken together, these data are consistent with an elongated beads-on-a-string arrangement of the tandem ACP domains in PUFA synthases, and speak against synergistic biocatalytic effects promoted by quaternary structuring. Thus, it is possible to envision bioengineering strategies which simply involve the artificial linking of multiple ACP domains for increasing the yield of fatty acids in bacterial cultures.


Subject(s)
Acyl Carrier Protein/chemistry , Fatty Acid Synthases/chemistry , Algorithms , Amino Acid Sequence , Models, Molecular , Molecular Sequence Data , Peptide Fragments/chemistry , Photobacterium/enzymology , Protein Denaturation , Protein Structure, Tertiary , Solutions , Temperature
7.
Cell ; 149(7): 1514-24, 2012 Jun 22.
Article in English | MEDLINE | ID: mdl-22726438

ABSTRACT

Receptor tyrosine kinase activity is known to occur in the absence of extracellular stimuli. Importantly, this "background" level of receptor phosphorylation is insufficient to effect a downstream response, suggesting that strict controls are present and prohibit full activation. Here a mechanism is described in which control of FGFR2 activation is provided by the adaptor protein Grb2. Dimeric Grb2 binds to the C termini of two FGFR2 molecules. This heterotetramer is capable of a low-level receptor transphosphorylation, but C-terminal phosphorylation and recruitment of signaling proteins are sterically hindered. Upon stimulation, FGFR2 phosphorylates tyrosine residues on Grb2, promoting dissociation from the receptor and allowing full activation of downstream signaling. These observations establish a role for Grb2 as an active regulator of RTK signaling.


Subject(s)
GRB2 Adaptor Protein/metabolism , Receptor, Fibroblast Growth Factor, Type 2/metabolism , Signal Transduction , Dimerization , HEK293 Cells , Humans , Models, Molecular , Phosphorylation , Receptor, Fibroblast Growth Factor, Type 2/chemistry
8.
J Am Chem Soc ; 134(24): 10138-45, 2012 Jun 20.
Article in English | MEDLINE | ID: mdl-22621321

ABSTRACT

In this study, we present advances in the use of rhodium(II) metallopeptides for protein modification. Site-specific, proximity-driven modification is enabled by the unique combination of peptide-based molecular recognition and a rhodium catalyst capable of modifying a wide range of amino-acid side chains. We explore catalysis based on coiled-coil recognition in detail, providing an understanding of the determinants of specificity and culminating in the demonstration of orthogonal modification of separate proteins in cell lysate. In addition, the concepts of proximity-driven catalysis are extended to include modification of the natural Fyn SH3 domain with metallopeptides based on a known proline-rich peptide ligand. The development of orthogonal catalyst-substrate pairs for modification in lysate, and the extension of these methods to new natural protein domains, highlight the capabilities for new reaction design possible in chemical approaches to site-specific protein modification.


Subject(s)
Organometallic Compounds/chemistry , Peptides/chemistry , Proteins/chemistry , Rhodium/chemistry , Amino Acid Sequence , Catalysis , Humans , Models, Molecular , Molecular Sequence Data , Organometallic Compounds/metabolism , Peptides/metabolism , Proteins/metabolism , Proto-Oncogene Proteins c-fyn/chemistry , Proto-Oncogene Proteins c-fyn/metabolism , Rhodium/metabolism , src Homology Domains
9.
J Biol Chem ; 287(1): 736-747, 2012 Jan 02.
Article in English | MEDLINE | ID: mdl-22102410

ABSTRACT

Ample evidence suggests that almost all polypeptides can either adopt a native structure (folded or intrinsically disordered) or form misfolded amyloid fibrils. Soluble protein oligomers exist as an intermediate between these two states, and their cytotoxicity has been implicated in the pathology of multiple human diseases. However, the mechanism by which soluble protein oligomers develop into insoluble amyloid fibrils is not clear, and investigation of this important issue is hindered by the unavailability of stable protein oligomers. Here, we have obtained stabilized protein oligomers generated from common native proteins. These oligomers exert strong cytotoxicity and display a common conformational structure shared with known protein oligomers. They are soluble and remain stable in solution. Intriguingly, the stabilized protein oligomers interact preferentially with both nucleic acids and glycosaminoglycans (GAG), which facilitates their rapid conversion into insoluble amyloid. Concomitantly, binding with nucleic acids or GAG strongly diminished the cytotoxicity of the protein oligomers. EGCG, a small molecule that was previously shown to directly bind to protein oligomers, effectively inhibits the conversion to amyloid. These results indicate that stabilized oligomers of common proteins display characteristics similar to those of disease-associated protein oligomers and represent immediate precursors of less toxic amyloid fibrils. Amyloid conversion is potently expedited by certain physiological factors, such as nucleic acids and GAGs. These findings concur with reports of cofactor involvement with disease-associated amyloid and shed light on potential means to interfere with the pathogenic properties of misfolded proteins.


Subject(s)
Amyloidogenic Proteins/chemistry , Amyloidogenic Proteins/metabolism , DNA/metabolism , Glycosaminoglycans/metabolism , Protein Multimerization , RNA/metabolism , Amyloidogenic Proteins/toxicity , Carbodiimides/pharmacology , Catechin/analogs & derivatives , Catechin/pharmacology , Cell Death/drug effects , Cross-Linking Reagents/pharmacology , HEK293 Cells , Humans , Jurkat Cells , Methylamines/pharmacology , Protein Multimerization/drug effects , Protein Stability , Protein Structure, Quaternary , Solubility
10.
J Biol Chem ; 286(8): 6685-96, 2011 Feb 25.
Article in English | MEDLINE | ID: mdl-21156796

ABSTRACT

The inhibitory T-cell surface-expressed receptor, cytotoxic T lymphocyte-associated antigen-4 (CTLA-4), which belongs to the class of cell surface proteins phosphorylated by extrinsic tyrosine kinases that also includes antigen receptors, binds the related ligands, B7-1 and B7-2, expressed on antigen-presenting cells. Conformational changes are commonly invoked to explain ligand-induced "triggering" of this class of receptors. Crystal structures of ligand-bound CTLA-4 have been reported, but not the apo form, precluding analysis of the structural changes accompanying ligand binding. The 1.8-Å resolution structure of an apo human CTLA-4 homodimer emphasizes the shared evolutionary history of the CTLA-4/CD28 subgroup of the immunoglobulin superfamily and the antigen receptors. The ligand-bound and unbound forms of both CTLA-4 and B7-1 are remarkably similar, in marked contrast to B7-2, whose binding to CTLA-4 has elements of induced fit. Isothermal titration calorimetry reveals that ligand binding by CTLA-4 is enthalpically driven and accompanied by unfavorable entropic changes. The similarity of the thermodynamic parameters determined for the interactions of CTLA-4 with B7-1 and B7-2 suggests that the binding is not highly specific, but the conformational changes observed for B7-2 binding suggest some level of selectivity. The new structure establishes that rigid-body ligand interactions are capable of triggering CTLA-4 phosphorylation by extrinsic kinase(s).


Subject(s)
Antigens, CD/chemistry , B7-1 Antigen/chemistry , B7-2 Antigen/chemistry , Receptors, Antigen, T-Cell/chemistry , Animals , Antigens, CD/genetics , Antigens, CD/immunology , B7-1 Antigen/genetics , B7-1 Antigen/immunology , B7-2 Antigen/genetics , B7-2 Antigen/immunology , Binding Sites , CHO Cells , CTLA-4 Antigen , Cricetinae , Cricetulus , Crystallography, X-Ray , Humans , Protein Structure, Tertiary , Receptors, Antigen, T-Cell/genetics , Receptors, Antigen, T-Cell/immunology , Thermodynamics
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