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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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Comparison of theoretical isoelectric points (pIs) ofproteinsdetected in the protein-NP complex derived from OSE or A2780 lysates.Proteins that are attached at any time point (a,d) have higher averagepI compared to respective lysate pools. Similarly, proteins that adsorbto AuNPs exclusively at shorter time points also have higher pIs (b,e)along with core proteins that are always present in the protein-NPcomplex (c,f). Red line represents the average pI. (Tukey’smultiple comparison test, unpaired t test *P ≤ 0.05, ** P ≤ 0.01, ***P < 0.001).
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fig5: Comparison of theoretical isoelectric points (pIs) ofproteinsdetected in the protein-NP complex derived from OSE or A2780 lysates.Proteins that are attached at any time point (a,d) have higher averagepI compared to respective lysate pools. Similarly, proteins that adsorbto AuNPs exclusively at shorter time points also have higher pIs (b,e)along with core proteins that are always present in the protein-NPcomplex (c,f). Red line represents the average pI. (Tukey’smultiple comparison test, unpaired t test *P ≤ 0.05, ** P ≤ 0.01, ***P < 0.001).

Mentions: To understandthe interaction of proteins and AuNPs, we examined various characteristicsof the detected proteins that were attached to the AuNPs. Proteinsthat are bound to NPs at multiple time points had a significantlyhigher mean theoretical isoelectric points (pI) (7.5, 7.5, 7.7, 7.7,and 7.7 for OSE; 7.5, 7.8, 7.0, and 7.5 for A2780) compared to thelysate pools (6.5 for OSE; 6.3 for A2780) (Figure 5a,d). The proteins that were present in the NP-protein complexat all the time points also had a similar correlation with pI, whereattached proteins had significantly higher pIs (Figure 5c,f). The mean pI of attached OSE and A2780 proteins was 7.7and 7.6, respectively, whereas pI of OSE and A2780 pooled lysateswas 6.5 and 6.3. Interestingly, proteins that were bound exclusivelyat 5 and 15 min had higher pIs. In the case of OSE proteins, thoseattached at 5 and 15 min had an average pI of 8.3 and 8.1, and forA2780 proteins, the pI at those time points was 7.4 and 7.6 (Figure 5b,e). This observation suggested that electrostaticinteraction played an important role in NP–protein interactionat initial time points. The charge of the protein as one of the contributingfactors influencing adsorption of proteins to AuNPs was in agreementwith a previous study that reported that BSA must interact with citrate-coatedAuNPs via salt-bridges possibly between citrate and lysine residueson the protein surface.31 We also examinedthe molecular weights (MWs) of proteins that attached to AuNPs andfound no difference in the average MW of proteins that are attachedvs the lysate pool (Supporting Information, Figure S6).


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)

Comparison of theoretical isoelectric points (pIs) ofproteinsdetected in the protein-NP complex derived from OSE or A2780 lysates.Proteins that are attached at any time point (a,d) have higher averagepI compared to respective lysate pools. Similarly, proteins that adsorbto AuNPs exclusively at shorter time points also have higher pIs (b,e)along with core proteins that are always present in the protein-NPcomplex (c,f). Red line represents the average pI. (Tukey’smultiple comparison test, unpaired t test *P ≤ 0.05, ** P ≤ 0.01, ***P < 0.001).
© Copyright Policy
Related In: Results  -  Collection

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

fig5: Comparison of theoretical isoelectric points (pIs) ofproteinsdetected in the protein-NP complex derived from OSE or A2780 lysates.Proteins that are attached at any time point (a,d) have higher averagepI compared to respective lysate pools. Similarly, proteins that adsorbto AuNPs exclusively at shorter time points also have higher pIs (b,e)along with core proteins that are always present in the protein-NPcomplex (c,f). Red line represents the average pI. (Tukey’smultiple comparison test, unpaired t test *P ≤ 0.05, ** P ≤ 0.01, ***P < 0.001).
Mentions: To understandthe interaction of proteins and AuNPs, we examined various characteristicsof the detected proteins that were attached to the AuNPs. Proteinsthat are bound to NPs at multiple time points had a significantlyhigher mean theoretical isoelectric points (pI) (7.5, 7.5, 7.7, 7.7,and 7.7 for OSE; 7.5, 7.8, 7.0, and 7.5 for A2780) compared to thelysate pools (6.5 for OSE; 6.3 for A2780) (Figure 5a,d). The proteins that were present in the NP-protein complexat all the time points also had a similar correlation with pI, whereattached proteins had significantly higher pIs (Figure 5c,f). The mean pI of attached OSE and A2780 proteins was 7.7and 7.6, respectively, whereas pI of OSE and A2780 pooled lysateswas 6.5 and 6.3. Interestingly, proteins that were bound exclusivelyat 5 and 15 min had higher pIs. In the case of OSE proteins, thoseattached at 5 and 15 min had an average pI of 8.3 and 8.1, and forA2780 proteins, the pI at those time points was 7.4 and 7.6 (Figure 5b,e). This observation suggested that electrostaticinteraction played an important role in NP–protein interactionat initial time points. The charge of the protein as one of the contributingfactors influencing adsorption of proteins to AuNPs was in agreementwith a previous study that reported that BSA must interact with citrate-coatedAuNPs via salt-bridges possibly between citrate and lysine residueson the protein surface.31 We also examinedthe molecular weights (MWs) of proteins that attached to AuNPs andfound no difference in the average MW of proteins that are attachedvs the lysate pool (Supporting Information, Figure S6).

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