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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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(a) Expression of PPA1, SMNDC1, and PI15in various ovarian cancercell lines and normal OSE cells as determined through immunoblottinganalysis with actin as loading control. (b) Effect of siRNA mediatedsilencing on the proliferation of A2780 cells determined by 3H-thymidine incorporation assay. (c) Immunoblot analysis to confirmefficient knockdown of the targets. Actin is used as the loading control.
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fig9: (a) Expression of PPA1, SMNDC1, and PI15in various ovarian cancercell lines and normal OSE cells as determined through immunoblottinganalysis with actin as loading control. (b) Effect of siRNA mediatedsilencing on the proliferation of A2780 cells determined by 3H-thymidine incorporation assay. (c) Immunoblot analysis to confirmefficient knockdown of the targets. Actin is used as the loading control.

Mentions: To validate our bioinformatics-based network analyses andto demonstratethe biological significance of hub proteins, we studied the proteinexpression of three nodal proteins that have variable connectivity,namely, PPA1 [Pyrophosphatase (Inorganic) 1], SMNDC1 (Survival MotorNeuron Domain Containing 1), and PI15 (Peptidase Inhibitor 15). SMNDC1was one of the top nodes detected at both 6 and 24 h. PPA1 and PI15,on the other hand, were only detected at 6 h with the former displayingmultiple protein connections while the latter was limited to one.Despite their connectivity status all three proteins were detectedwith the help of NPs from A2780 cell lysates only. Furthermore, thebiological function of these selected hub proteins in ovarian canceris currently unknown. Functionally, PPA1 catalyzes the hydrolysisof pyrophosphate to inorganic phosphate, which is important for thephosphate metabolism of cells. There is only a single report indicatinga role of PPA1 in pathogenesis of gastric cancer.42 SMNDC1 is a nuclear protein that has been identified asa constituent of the spliceosome complex and has been reported topossess anti-apoptotic function together with Bcl-2. Loss of its paralog,SMN, in spinal muscular atrophy has thus been suggested to be involvedin the pathogenesis of the disease.43 However,any role of SMNDC1 in cancer has not been defined so far. PI15 belongsto the family of trypsin inhibitors and the role of this class ofproteases in gynecological cancers have been reported, but detailedmechanistic studies and therapeutic strategies to inhibit their functionare currently lacking.44 Lastly, PI15 hasbeen detected abundantly in human neuroblastoma and glioblastoma celllines.45 We examined expression of allthree proteins in a panel of ovarian cancer cell lines and comparedthe levels with normal OSE cell line. Western blot analysis showedrelative overexpression in most ovarian cancer cells in comparisonto normal OSE cells (Figure 9a) which explainstheir enrichment from A2780 lysates.


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)

(a) Expression of PPA1, SMNDC1, and PI15in various ovarian cancercell lines and normal OSE cells as determined through immunoblottinganalysis with actin as loading control. (b) Effect of siRNA mediatedsilencing on the proliferation of A2780 cells determined by 3H-thymidine incorporation assay. (c) Immunoblot analysis to confirmefficient knockdown of the targets. Actin is used as the loading control.
© Copyright Policy
Related In: Results  -  Collection

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

fig9: (a) Expression of PPA1, SMNDC1, and PI15in various ovarian cancercell lines and normal OSE cells as determined through immunoblottinganalysis with actin as loading control. (b) Effect of siRNA mediatedsilencing on the proliferation of A2780 cells determined by 3H-thymidine incorporation assay. (c) Immunoblot analysis to confirmefficient knockdown of the targets. Actin is used as the loading control.
Mentions: To validate our bioinformatics-based network analyses andto demonstratethe biological significance of hub proteins, we studied the proteinexpression of three nodal proteins that have variable connectivity,namely, PPA1 [Pyrophosphatase (Inorganic) 1], SMNDC1 (Survival MotorNeuron Domain Containing 1), and PI15 (Peptidase Inhibitor 15). SMNDC1was one of the top nodes detected at both 6 and 24 h. PPA1 and PI15,on the other hand, were only detected at 6 h with the former displayingmultiple protein connections while the latter was limited to one.Despite their connectivity status all three proteins were detectedwith the help of NPs from A2780 cell lysates only. Furthermore, thebiological function of these selected hub proteins in ovarian canceris currently unknown. Functionally, PPA1 catalyzes the hydrolysisof pyrophosphate to inorganic phosphate, which is important for thephosphate metabolism of cells. There is only a single report indicatinga role of PPA1 in pathogenesis of gastric cancer.42 SMNDC1 is a nuclear protein that has been identified asa constituent of the spliceosome complex and has been reported topossess anti-apoptotic function together with Bcl-2. Loss of its paralog,SMN, in spinal muscular atrophy has thus been suggested to be involvedin the pathogenesis of the disease.43 However,any role of SMNDC1 in cancer has not been defined so far. PI15 belongsto the family of trypsin inhibitors and the role of this class ofproteases in gynecological cancers have been reported, but detailedmechanistic studies and therapeutic strategies to inhibit their functionare currently lacking.44 Lastly, PI15 hasbeen detected abundantly in human neuroblastoma and glioblastoma celllines.45 We examined expression of allthree proteins in a panel of ovarian cancer cell lines and comparedthe levels with normal OSE cell line. Western blot analysis showedrelative overexpression in most ovarian cancer cells in comparisonto normal OSE cells (Figure 9a) which explainstheir enrichment from A2780 lysates.

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