9 results on '"Johnson, Jennifer M."'
Search Results
2. Redirecting the coat protein of a spherical virus to assemble into tubular nanostructures
- Author
-
Mukherjee, Santanu, Pfeifer, Cory M., Johnson, Jennifer M., Liu, Jay, and Zlotnick, Adam
- Subjects
Nanotechnology -- Research ,Proteins -- Chemical properties ,Nanotubes -- Research ,Chemistry - Abstract
A study aims at assembling a structure that can be used as a platform for building more complex nanostructures. An application of virus capsid proteins in generating structures that are a highly organized scaffold from which more complex networks may be prepared where the tunable length distribution of the coat protein nanotubes contributes to this utility.
- Published
- 2006
3. Conformation-Dependent Human p52Shc Phosphorylation by Human c-Src.
- Author
-
Yuko Tsutsui, Johnson, Jennifer M., Demeler, Borries, Kinter, Michael T., and Hays, Franklin A.
- Subjects
- *
PHOSPHORYLATION , *ADAPTOR proteins , *PROTEIN-tyrosine kinases , *MASS spectrometry , *PHOSPHATIDYLINOSITOL 3-kinases - Abstract
Phosphorylation of the human p52Shc adaptor protein is a key determinant in modulating signaling complex assembly in response to tyrosine kinase signaling cascade activation. The underlying mechanisms that govern p52Shc phosphorylation status are unknown. In this study, p52Shc phosphorylation by human c-Src was investigated using purified proteins to define mechanisms that affect the p52Shc phosphorylation state. We conducted biophysical characterizations of both human p52Shc and human c-Src in solution as well as membrane-mimetic environments using the acidic lipid phosphatidylinositol 4-phosphate or a novel amphipathic detergent (2,2-dihexylpropane-1,3-bis-β-D-glucopyranoside). We then identified p52Shc phosphorylation sites under various solution conditions, and the amount of phosphorylation at each identified site was quantified using mass spectrometry. These data demonstrate that the p52Shc phosphorylation level is altered by the solution environment without affecting the fraction of active c-Src. Mass spectrometry analysis of phosphorylated p52Shc implies functional linkage among phosphorylation sites. This linkage may drive preferential coupling to protein binding partners during signaling complex formation, such as during initial binding interactions with the Grb2 adaptor protein leading to activation of the Ras/MAPK signaling cascade. Remarkably, tyrosine residues involved in Grb2 binding were heavily phosphorylated in a membrane-mimetic environment. The increased phosphorylation level in Grb2 binding residues was also correlated with a decrease in the thermal stability of purified human p52Shc. A schematic for the phosphorylation-dependent interaction between p52Shc and Grb2 is proposed. The results of this study suggest another possible therapeutic strategy for altering protein phosphorylation to regulate signaling cascade activation. [ABSTRACT FROM AUTHOR]
- Published
- 2015
- Full Text
- View/download PDF
4. Requirements for Skp1 Processing by Cytosolic Prolyl 4(trαns)-Hydroxylαse αnd α-N-αcetylglucosαminyltrαnsferαse Enzymes Involved in O2 Signαling in Dictyostelium.
- Author
-
van der Wel, Hanke, Johnson, Jennifer M., Yuechi Xu, Karunaratne, Chamini V., Wilson, Kyle D., Vohra, Yusuf, Boons, Geert-Jan, Taylor, Carol M., Bendiak, Brad, and West, Christopher M.
- Subjects
- *
PEPTIDYLPROLYL isomerase , *DICTYOSTELIUM , *HYPOXIA-inducible factors , *UBIQUITIN , *KREBS cycle , *HYDROXYLATION - Abstract
The social amoeba Dictyostelium expresses a hypoxia inducible factor-α (HIFα) type prolyl 4-hydroxylase (P4H1) and an a-N-acetylglucosaminyltransferase (Gnt1) that sequentially modify proline-143 of Skp1, a subunit of the SCF (Skp1/Cullin/F-box protein) class of E3 ubiquitin ligases. Prior genetic studies have implicated Skp1 and its modification by these enzymes in O2 regulation of development, suggesting the existence of an ancient O2-sensing mechanism related to modification of the transcription factor HIFa by animal prolyl 4-hydroxylases (PHDs). To better understand the role of Skp1 in P4H1-dependent O2 signaling, biochemical and biophysical studies were conducted to characterize the reaction product and the basis of Skp1 substrate selection by P4H1 and Gnt1. 1H NMR demonstrated formation of 4(trans)-hydroxyproline as previously found for HIFα, and highly purified P4H1 was inhibited by Krebs cycle intermediates and other compounds that affect animal P4Hs. However, in contrast to hydroxylation of HIFa by PHDs, P4H1 depended on features of full-length Skp1, based on truncation, mutagenesis, and competitive inhibition studies. These features are conserved during animal evolution, as even mammalian Skp1, which lacks the target proline, became a good substrate upon its restoration. P4H1 recognition may depend on features conserved for SCF complex formation as heterodimerization with an F-box protein blocked Skp1 hydroxylation. The hydroxyproline-capping enzyme Gnt1 exhibited similar requirements for Skp1 as a substrate. These and other findings support a model in which the protist P4H1 conditionally hydroxylates Skp1 of E3SCFubiquitin ligases to control half-lives of multiple targets, rather than the mechanism of animal PHDs where individual proteins are hydroxylated leading to ubiquitination by the evolutionarily related E3VBCubiquitin ligases. [ABSTRACT FROM AUTHOR]
- Published
- 2011
- Full Text
- View/download PDF
5. Conformation-Dependent Human p52Shc Phosphorylation by Human c-Src.
- Author
-
Tsutsui Y, Johnson JM, Demeler B, Kinter MT, and Hays FA
- Subjects
- CSK Tyrosine-Protein Kinase, Cell Membrane chemistry, Cell Membrane genetics, Extracellular Signal-Regulated MAP Kinases chemistry, Extracellular Signal-Regulated MAP Kinases genetics, Extracellular Signal-Regulated MAP Kinases metabolism, GRB2 Adaptor Protein chemistry, GRB2 Adaptor Protein genetics, GRB2 Adaptor Protein metabolism, Humans, MAP Kinase Signaling System physiology, Phosphatidylinositol Phosphates chemistry, Phosphorylation physiology, Protein Stability, Proto-Oncogene Proteins p21(ras) chemistry, Proto-Oncogene Proteins p21(ras) genetics, Proto-Oncogene Proteins p21(ras) metabolism, Shc Signaling Adaptor Proteins chemistry, Shc Signaling Adaptor Proteins genetics, Src Homology 2 Domain-Containing, Transforming Protein 1, src-Family Kinases genetics, Cell Membrane metabolism, Shc Signaling Adaptor Proteins metabolism, src-Family Kinases chemistry, src-Family Kinases metabolism
- Abstract
Phosphorylation of the human p52Shc adaptor protein is a key determinant in modulating signaling complex assembly in response to tyrosine kinase signaling cascade activation. The underlying mechanisms that govern p52Shc phosphorylation status are unknown. In this study, p52Shc phosphorylation by human c-Src was investigated using purified proteins to define mechanisms that affect the p52Shc phosphorylation state. We conducted biophysical characterizations of both human p52Shc and human c-Src in solution as well as membrane-mimetic environments using the acidic lipid phosphatidylinositol 4-phosphate or a novel amphipathic detergent (2,2-dihexylpropane-1,3-bis-β-D-glucopyranoside). We then identified p52Shc phosphorylation sites under various solution conditions, and the amount of phosphorylation at each identified site was quantified using mass spectrometry. These data demonstrate that the p52Shc phosphorylation level is altered by the solution environment without affecting the fraction of active c-Src. Mass spectrometry analysis of phosphorylated p52Shc implies functional linkage among phosphorylation sites. This linkage may drive preferential coupling to protein binding partners during signaling complex formation, such as during initial binding interactions with the Grb2 adaptor protein leading to activation of the Ras/MAPK signaling cascade. Remarkably, tyrosine residues involved in Grb2 binding were heavily phosphorylated in a membrane-mimetic environment. The increased phosphorylation level in Grb2 binding residues was also correlated with a decrease in the thermal stability of purified human p52Shc. A schematic for the phosphorylation-dependent interaction between p52Shc and Grb2 is proposed. The results of this study suggest another possible therapeutic strategy for altering protein phosphorylation to regulate signaling cascade activation.
- Published
- 2015
- Full Text
- View/download PDF
6. Requirements for Skp1 processing by cytosolic prolyl 4(trans)-hydroxylase and α-N-acetylglucosaminyltransferase enzymes involved in O₂ signaling in dictyostelium.
- Author
-
van der Wel H, Johnson JM, Xu Y, Karunaratne CV, Wilson KD, Vohra Y, Boons GJ, Taylor CM, Bendiak B, and West CM
- Subjects
- Animals, Models, Molecular, Protein Binding, Protein Structure, Tertiary, SKP Cullin F-Box Protein Ligases chemistry, Substrate Specificity, Cytosol enzymology, Dictyostelium enzymology, N-Acetylglucosaminyltransferases metabolism, Oxygen metabolism, Procollagen-Proline Dioxygenase metabolism, SKP Cullin F-Box Protein Ligases metabolism, Signal Transduction
- Abstract
The social amoeba Dictyostelium expresses a hypoxia inducible factor-α (HIFα) type prolyl 4-hydroxylase (P4H1) and an α-N-acetylglucosaminyltransferase (Gnt1) that sequentially modify proline-143 of Skp1, a subunit of the SCF (Skp1/Cullin/F-box protein) class of E3 ubiquitin ligases. Prior genetic studies have implicated Skp1 and its modification by these enzymes in O(2) regulation of development, suggesting the existence of an ancient O(2)-sensing mechanism related to modification of the transcription factor HIFα by animal prolyl 4-hydroxylases (PHDs). To better understand the role of Skp1 in P4H1-dependent O(2) signaling, biochemical and biophysical studies were conducted to characterize the reaction product and the basis of Skp1 substrate selection by P4H1 and Gnt1. (1)H NMR demonstrated formation of 4(trans)-hydroxyproline as previously found for HIFα, and highly purified P4H1 was inhibited by Krebs cycle intermediates and other compounds that affect animal P4Hs. However, in contrast to hydroxylation of HIFα by PHDs, P4H1 depended on features of full-length Skp1, based on truncation, mutagenesis, and competitive inhibition studies. These features are conserved during animal evolution, as even mammalian Skp1, which lacks the target proline, became a good substrate upon its restoration. P4H1 recognition may depend on features conserved for SCF complex formation as heterodimerization with an F-box protein blocked Skp1 hydroxylation. The hydroxyproline-capping enzyme Gnt1 exhibited similar requirements for Skp1 as a substrate. These and other findings support a model in which the protist P4H1 conditionally hydroxylates Skp1 of E3(SCF)ubiquitin ligases to control half-lives of multiple targets, rather than the mechanism of animal PHDs where individual proteins are hydroxylated leading to ubiquitination by the evolutionarily related E3(VBC)ubiquitin ligases.
- Published
- 2011
- Full Text
- View/download PDF
7. Glycopeptidome of a heavily N-glycosylated cell surface glycoprotein of Dictyostelium implicated in cell adhesion.
- Author
-
Feasley CL, Johnson JM, West CM, and Chia CP
- Subjects
- Amino Acid Sequence, Base Sequence, Chromatography, High Pressure Liquid, Electrophoresis, Polyacrylamide Gel, Glycomics, Glycopeptides metabolism, Glycosylation, Membrane Glycoproteins genetics, Membrane Glycoproteins metabolism, Molecular Sequence Data, Polysaccharides, Protozoan Proteins genetics, Protozoan Proteins metabolism, Tandem Mass Spectrometry, Cell Adhesion, Dictyostelium genetics, Glycopeptides chemistry, Membrane Glycoproteins chemistry, Protozoan Proteins chemistry
- Abstract
Genetic analysis has implicated the cell surface glycoprotein gp130 in cell interactions of the social amoeba Dictyostelium, and information about the utilization of the 18 N-glycosylation sequons present in gp130 is needed to identify critical molecular determinants of its activity. Various glycomics strategies, including mass spectrometry of native and derivatized glycans, monosaccharide analysis, exoglycosidase digestion, and antibody binding, were applied to characterize a nonanchored version secreted from Dictyostelium. s-gp130 is modified by a predominant Man(8)GlcNAc(4) species containing bisecting and intersecting GlcNAc residues and additional high-mannose N-glycans substituted with sulfate, methyl-phosphate, and/or core alpha 3-fucose. Site mapping confirmed the occupancy of 15 sequons, some variably, and glycopeptide analysis confirmed 14 sites and revealed extensive heterogeneity at most sites. Glycopeptide glycoforms ranged from Man(6) to Man(9), GlcNAc(0-2) (peripheral), Fuc(0-2) (including core alpha 3 and peripheral), (SO(4))(0-1), and (MePO(4))(0-1), which represented elements of virtually the entire known cellular N-glycome as inferred from prior metabolic labeling and mass spectrometry studies. gp130, and a family of 14 related predicted glycoproteins whose polypeptide sequences are rapidly diverging in the Dictyostelium lineage, may contribute a functionally important shroud of high-mannose N-glycans at the interface of the amoebae with each other, their predators and prey, and the soil environment.
- Published
- 2010
- Full Text
- View/download PDF
8. Altering the energy landscape of virus self-assembly to generate kinetically trapped nanoparticles.
- Author
-
Burns K, Mukherjee S, Keef T, Johnson JM, and Zlotnick A
- Subjects
- Capsid Proteins chemistry, Capsid Proteins physiology, Energy Transfer physiology, Surface Properties, Bromovirus chemistry, Bromovirus physiology, Nanoparticles chemistry, Virus Assembly physiology
- Abstract
Controlling self-assembly is critical to the advancement of nanotechnology. A rugged or crenated assembly energy surface can redirect assembly off path. By using a defined starting point and an energy surface made rough by a strong association energy, we can impose entirely new assembly paths and products. Normally, the coat protein (CP) of the Cowpea Chlorotic Mottle Virus (CCMV) assembles into virus-like 28 nm diameter icosahedral particles. Here we have started with the coat protein trapped in a rod-like structure in complex with DNA. When these 17 nm diameter rods are placed under the same condition, low pH, that normally leads to assembly of 28 nm diameter particles, we instead obtain 17 nm capsids. The extrusion of all-pentamer capsids from the hexagonal lattice of the rod demonstrates the importance of the starting state for controlled assembly.
- Published
- 2010
- Full Text
- View/download PDF
9. Regulating self-assembly of spherical oligomers.
- Author
-
Johnson JM, Tang J, Nyame Y, Willits D, Young MJ, and Zlotnick A
- Subjects
- Kinetics, Models, Chemical, Bromovirus chemistry, Capsid Proteins chemistry, Virus Assembly
- Abstract
In multistep reactions, stability of intermediates is critical to the rate of product formation and a significant factor in generating kinetic traps. The capsid protein of cowpea chlorotic mottle virus (CCMV) can be induced to assemble into spherical particles of 30, 60, and 90 dimers. Based on examining assembly kinetics and reaction end points, we find that formation of uniform, ordered structures is not always a result of reactions that reach equilibrium. Equilibration or, alternatively, kinetic trapping can be identified by a straightforward analysis. Altering the assembly path of "spherical" particles is a means of controlling the distribution of products, which has broad applicability to self-assembly reactions.
- Published
- 2005
- Full Text
- View/download PDF
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