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Understanding protein-nanoparticle interaction: a new gateway to disease therapeutics.

Giri K, Shameer K, Zimmermann MT, Saha S, Chakraborty PK, Sharma A, Arvizo RR, Madden BJ, Mccormick DJ, Kocher JP, Bhattacharya R, Mukherjee P - Bioconjug. Chem. (2014)

Bottom Line: The importance of this methodology and the biological significance of the network proteins were validated by a functional study of three hubs that exhibited variable connectivity, namely, PPA1, SMNDC1, and PI15.Western blot analysis revealed overexpression of these proteins in ovarian cancer cells when compared to normal cells.Silencing of PPA1, SMNDC1, and PI15 by the siRNA approach significantly inhibited proliferation of ovarian cancer cells and the effect correlated with the connectivity pattern obtained from our network analyses.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry and Molecular Biology, ‡Division of Biomedical Statistics and Informatics, Department of Health Sciences Research, ⊥Molecular Medicine Program, and #Proteomics Research Center, Mayo Clinic , Rochester, Minnesota 55905, United States.

ABSTRACT
Molecular identification of protein molecules surrounding nanoparticles (NPs) may provide useful information that influences NP clearance, biodistribution, and toxicity. Hence, nanoproteomics provides specific information about the environment that NPs interact with and can therefore report on the changes in protein distribution that occurs during tumorigenesis. Therefore, we hypothesized that characterization and identification of protein molecules that interact with 20 nm AuNPs from cancer and noncancer cells may provide mechanistic insights into the biology of tumor growth and metastasis and identify new therapeutic targets in ovarian cancer. Hence, in the present study, we systematically examined the interaction of the protein molecules with 20 nm AuNPs from cancer and noncancerous cell lysates. Time-resolved proteomic profiles of NP-protein complexes demonstrated electrostatic interaction to be the governing factor in the initial time-points which are dominated by further stabilization interaction at longer time-points as determined by ultraviolet-visible spectroscopy (UV-vis), dynamic light scattering (DLS), ζ-potential measurements, transmission electron microscopy (TEM), and tandem mass spectrometry (MS/MS). Reduction in size, charge, and number of bound proteins were observed as the protein-NP complex stabilized over time. Interestingly, proteins related to mRNA processing were overwhelmingly represented on the NP-protein complex at all times. More importantly, comparative proteomic analyses revealed enrichment of a number of cancer-specific proteins on the AuNP surface. Network analyses of these proteins highlighted important hub nodes that could potentially be targeted for maximal therapeutic advantage in the treatment of ovarian cancer. The importance of this methodology and the biological significance of the network proteins were validated by a functional study of three hubs that exhibited variable connectivity, namely, PPA1, SMNDC1, and PI15. Western blot analysis revealed overexpression of these proteins in ovarian cancer cells when compared to normal cells. Silencing of PPA1, SMNDC1, and PI15 by the siRNA approach significantly inhibited proliferation of ovarian cancer cells and the effect correlated with the connectivity pattern obtained from our network analyses.

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Visualization of protein layer aroundAuNPs with transmission electronmicroscopy after 24 h of incubation with A2780 lysates and negativestaining with phosphotungstic acid. Measurements were done with NPsthat were pelleted and washed once with water.
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fig3: Visualization of protein layer aroundAuNPs with transmission electronmicroscopy after 24 h of incubation with A2780 lysates and negativestaining with phosphotungstic acid. Measurements were done with NPsthat were pelleted and washed once with water.

Mentions: To gain more insight into theprotein-NP complex formation, wevisualized the biological layer around NPs after incubating with A2780protein lysate for 24 h by TEM (Figure 3).The NP bound proteins around NPs was negatively stained with phosphotungsticacid which revealed that the protein-NP complex could be asymmetricalinstead of being a uniformly distributed spherical layer around thespherical NPs. Some insoluble precipitates of phosphotungstic acidwere also present in the TEM grid which is typical of negative staining.30 Additionally, the TEM image suggested that NPscould come together to form doublets and act like a singular unitfor protein-NP complex formation. These results showcased the heterogeneityof the protein complex formation around NPs, the cause of which iscurrently under investigation.


Understanding protein-nanoparticle interaction: a new gateway to disease therapeutics.

Giri K, Shameer K, Zimmermann MT, Saha S, Chakraborty PK, Sharma A, Arvizo RR, Madden BJ, Mccormick DJ, Kocher JP, Bhattacharya R, Mukherjee P - Bioconjug. Chem. (2014)

Visualization of protein layer aroundAuNPs with transmission electronmicroscopy after 24 h of incubation with A2780 lysates and negativestaining with phosphotungstic acid. Measurements were done with NPsthat were pelleted and washed once with water.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4128259&req=5

fig3: Visualization of protein layer aroundAuNPs with transmission electronmicroscopy after 24 h of incubation with A2780 lysates and negativestaining with phosphotungstic acid. Measurements were done with NPsthat were pelleted and washed once with water.
Mentions: To gain more insight into theprotein-NP complex formation, wevisualized the biological layer around NPs after incubating with A2780protein lysate for 24 h by TEM (Figure 3).The NP bound proteins around NPs was negatively stained with phosphotungsticacid which revealed that the protein-NP complex could be asymmetricalinstead of being a uniformly distributed spherical layer around thespherical NPs. Some insoluble precipitates of phosphotungstic acidwere also present in the TEM grid which is typical of negative staining.30 Additionally, the TEM image suggested that NPscould come together to form doublets and act like a singular unitfor protein-NP complex formation. These results showcased the heterogeneityof the protein complex formation around NPs, the cause of which iscurrently under investigation.

Bottom Line: The importance of this methodology and the biological significance of the network proteins were validated by a functional study of three hubs that exhibited variable connectivity, namely, PPA1, SMNDC1, and PI15.Western blot analysis revealed overexpression of these proteins in ovarian cancer cells when compared to normal cells.Silencing of PPA1, SMNDC1, and PI15 by the siRNA approach significantly inhibited proliferation of ovarian cancer cells and the effect correlated with the connectivity pattern obtained from our network analyses.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry and Molecular Biology, ‡Division of Biomedical Statistics and Informatics, Department of Health Sciences Research, ⊥Molecular Medicine Program, and #Proteomics Research Center, Mayo Clinic , Rochester, Minnesota 55905, United States.

ABSTRACT
Molecular identification of protein molecules surrounding nanoparticles (NPs) may provide useful information that influences NP clearance, biodistribution, and toxicity. Hence, nanoproteomics provides specific information about the environment that NPs interact with and can therefore report on the changes in protein distribution that occurs during tumorigenesis. Therefore, we hypothesized that characterization and identification of protein molecules that interact with 20 nm AuNPs from cancer and noncancer cells may provide mechanistic insights into the biology of tumor growth and metastasis and identify new therapeutic targets in ovarian cancer. Hence, in the present study, we systematically examined the interaction of the protein molecules with 20 nm AuNPs from cancer and noncancerous cell lysates. Time-resolved proteomic profiles of NP-protein complexes demonstrated electrostatic interaction to be the governing factor in the initial time-points which are dominated by further stabilization interaction at longer time-points as determined by ultraviolet-visible spectroscopy (UV-vis), dynamic light scattering (DLS), ζ-potential measurements, transmission electron microscopy (TEM), and tandem mass spectrometry (MS/MS). Reduction in size, charge, and number of bound proteins were observed as the protein-NP complex stabilized over time. Interestingly, proteins related to mRNA processing were overwhelmingly represented on the NP-protein complex at all times. More importantly, comparative proteomic analyses revealed enrichment of a number of cancer-specific proteins on the AuNP surface. Network analyses of these proteins highlighted important hub nodes that could potentially be targeted for maximal therapeutic advantage in the treatment of ovarian cancer. The importance of this methodology and the biological significance of the network proteins were validated by a functional study of three hubs that exhibited variable connectivity, namely, PPA1, SMNDC1, and PI15. Western blot analysis revealed overexpression of these proteins in ovarian cancer cells when compared to normal cells. Silencing of PPA1, SMNDC1, and PI15 by the siRNA approach significantly inhibited proliferation of ovarian cancer cells and the effect correlated with the connectivity pattern obtained from our network analyses.

Show MeSH
Related in: MedlinePlus