Limits...
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.

Show MeSH

Related in: MedlinePlus

Enrichmentof proteins on AuNP surface and network properties ofproteins adsorbed to AuNPs at 6 and 24 h (a,b). Nodes are proteinsunique to A2780 protein-NP complex at the each time point, which werenot detected in the OSE or A2780 lysate pool. The size of networknodes indicates centrality measure derived from the functional networkat the given time points. Edge colors indicate the type of interactions:coexpression (violet), physical interaction (green), predicted interaction(blue), shared protein domains (yellow), or biological pathways (orange).Interactions were derived from GeneMania; network properties werecomputed using Cytoscape plugin Network Analyzer.
© Copyright Policy
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4128259&req=5

fig8: Enrichmentof proteins on AuNP surface and network properties ofproteins adsorbed to AuNPs at 6 and 24 h (a,b). Nodes are proteinsunique to A2780 protein-NP complex at the each time point, which werenot detected in the OSE or A2780 lysate pool. The size of networknodes indicates centrality measure derived from the functional networkat the given time points. Edge colors indicate the type of interactions:coexpression (violet), physical interaction (green), predicted interaction(blue), shared protein domains (yellow), or biological pathways (orange).Interactions were derived from GeneMania; network properties werecomputed using Cytoscape plugin Network Analyzer.

Mentions: We sought to utilizethe proteomic signature in the NP-protein complex to investigate thepotential use of AuNPs as a therapeutic target discovery tool. Wedecided to examine the NP-protein complex at 6 and 24 h because proteincharacterization and proteomic analyses demonstrated that the NP-proteincomplexes were stabilized at these time points. When comparing proteinsfrom the NP-protein complex at these time points and proteins detectedfrom the pooled lysate, we identified 41 and 65 A2780-specific proteinsthat were enriched on the NP surface at 6 and 24 h, respectively (Figure 8). Due to their differential expression in the A2780cell line, all of these proteins could be important for tumorigenicity.In addition, these proteins were not detected by doing mass spectrometryof the lysates perhaps because of their low abundance. The proteins’affinity to the AuNPs concentrated them on the surface for detection.Since probing individual proteins from the group is cumbersome andfraught with subjective interpretive problems, we utilized graph-theorybased network analyses algorithms to determine the connectivity ofthe proteins to each other and to identify key protein interactionnodes.38 Disabling one protein (node inthe network) that interacts with many others (high connectivity) maymaximize the therapeutic potential as it is already recognized thatdisrupting the function of a single protein is not sufficient to inhibittumor growth and metastasis.39,40 Figure 8 shows functional protein networks derived from cancer-specificproteins and the proteins were ranked according to the number of connectionsbased on coexpression, colocalization, physical interactions, andshared protein domains. ELF1AX, an essential eukaryotic translationinitiation factor, showed the highest degree of connectivity amongproteins enriched at 6 h. Other proteins identified with the highestnodal connections were PPA1, a member of inorganic pyrophosphatasefamily, SMNDC1, a survival motor neuron protein and PARK7, a memberof peptidase C56 family of proteins. In case of proteins enrichedat 24 h, the protein with the most connectivity was RPL12A, a ribosomal60s subunit protein. This finding was not surprising considering theenrichment of mRNA related protein on the NP surface. Others includedDEK, a DNA binding oncogene, DDX46, a probable ATP-dependent RNA helicase,and GNA13, a G-protein subunit. As evidenced by other analyses, mRNArelated proteins were highly enriched on the NP surface. While proteinssuch as ELF1AX and RPL10A,both related to protein translation, may be too broad to target andnonspecific for cancer cells (in spite of differential expression),others such as DEK, which displays oncogenic properties and regulateDNA damage response signaling,41 mightbe important to study in the context of ovarian cancer.


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)

Enrichmentof proteins on AuNP surface and network properties ofproteins adsorbed to AuNPs at 6 and 24 h (a,b). Nodes are proteinsunique to A2780 protein-NP complex at the each time point, which werenot detected in the OSE or A2780 lysate pool. The size of networknodes indicates centrality measure derived from the functional networkat the given time points. Edge colors indicate the type of interactions:coexpression (violet), physical interaction (green), predicted interaction(blue), shared protein domains (yellow), or biological pathways (orange).Interactions were derived from GeneMania; network properties werecomputed using Cytoscape plugin Network Analyzer.
© Copyright Policy
Related In: Results  -  Collection

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

fig8: Enrichmentof proteins on AuNP surface and network properties ofproteins adsorbed to AuNPs at 6 and 24 h (a,b). Nodes are proteinsunique to A2780 protein-NP complex at the each time point, which werenot detected in the OSE or A2780 lysate pool. The size of networknodes indicates centrality measure derived from the functional networkat the given time points. Edge colors indicate the type of interactions:coexpression (violet), physical interaction (green), predicted interaction(blue), shared protein domains (yellow), or biological pathways (orange).Interactions were derived from GeneMania; network properties werecomputed using Cytoscape plugin Network Analyzer.
Mentions: We sought to utilizethe proteomic signature in the NP-protein complex to investigate thepotential use of AuNPs as a therapeutic target discovery tool. Wedecided to examine the NP-protein complex at 6 and 24 h because proteincharacterization and proteomic analyses demonstrated that the NP-proteincomplexes were stabilized at these time points. When comparing proteinsfrom the NP-protein complex at these time points and proteins detectedfrom the pooled lysate, we identified 41 and 65 A2780-specific proteinsthat were enriched on the NP surface at 6 and 24 h, respectively (Figure 8). Due to their differential expression in the A2780cell line, all of these proteins could be important for tumorigenicity.In addition, these proteins were not detected by doing mass spectrometryof the lysates perhaps because of their low abundance. The proteins’affinity to the AuNPs concentrated them on the surface for detection.Since probing individual proteins from the group is cumbersome andfraught with subjective interpretive problems, we utilized graph-theorybased network analyses algorithms to determine the connectivity ofthe proteins to each other and to identify key protein interactionnodes.38 Disabling one protein (node inthe network) that interacts with many others (high connectivity) maymaximize the therapeutic potential as it is already recognized thatdisrupting the function of a single protein is not sufficient to inhibittumor growth and metastasis.39,40 Figure 8 shows functional protein networks derived from cancer-specificproteins and the proteins were ranked according to the number of connectionsbased on coexpression, colocalization, physical interactions, andshared protein domains. ELF1AX, an essential eukaryotic translationinitiation factor, showed the highest degree of connectivity amongproteins enriched at 6 h. Other proteins identified with the highestnodal connections were PPA1, a member of inorganic pyrophosphatasefamily, SMNDC1, a survival motor neuron protein and PARK7, a memberof peptidase C56 family of proteins. In case of proteins enrichedat 24 h, the protein with the most connectivity was RPL12A, a ribosomal60s subunit protein. This finding was not surprising considering theenrichment of mRNA related protein on the NP surface. Others includedDEK, a DNA binding oncogene, DDX46, a probable ATP-dependent RNA helicase,and GNA13, a G-protein subunit. As evidenced by other analyses, mRNArelated proteins were highly enriched on the NP surface. While proteinssuch as ELF1AX and RPL10A,both related to protein translation, may be too broad to target andnonspecific for cancer cells (in spite of differential expression),others such as DEK, which displays oncogenic properties and regulateDNA damage response signaling,41 mightbe important to study in the context of ovarian cancer.

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