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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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Analysis ofevolutionarily conserved functional domains (a) andbiological pathways (b) enriched in proteins detected at differenttime points revealed that AuNPs have a high affinity to mRNA relatedprotein machinery. Protein domains from Pfam database and pathwaysfrom databases KEGG (hsa*), Panther (P0*), and Reactome (REACT_*)that are significantly enriched among one or more lists are shown.Color of matrices indicates Bonferroni corrected P-values (P ≤ 0.05). Green indicates higherenrichment, red indicates lower enrichment, and gray color indicatesthat a given term did not reach statistical significance among theproteins in the list. Pathway enrichment analyses were performed usingDAVID v 6.1 using default settings. Human proteome was defined asthe background. (M = min, H = hours).
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fig6: Analysis ofevolutionarily conserved functional domains (a) andbiological pathways (b) enriched in proteins detected at differenttime points revealed that AuNPs have a high affinity to mRNA relatedprotein machinery. Protein domains from Pfam database and pathwaysfrom databases KEGG (hsa*), Panther (P0*), and Reactome (REACT_*)that are significantly enriched among one or more lists are shown.Color of matrices indicates Bonferroni corrected P-values (P ≤ 0.05). Green indicates higherenrichment, red indicates lower enrichment, and gray color indicatesthat a given term did not reach statistical significance among theproteins in the list. Pathway enrichment analyses were performed usingDAVID v 6.1 using default settings. Human proteome was defined asthe background. (M = min, H = hours).

Mentions: We next asked whether the proteins that were attached tothe NPsshared conserved domains32 which couldshed light on why some proteins adsorb onto the NP surface while othersdo not. Of the many domains that were enriched, from the proteinsbound to the NP surface RRM_1, a RNA recognition motif, was enrichedin proteins from all time point complexes (Figure 6a). The RRM_1 a motif is approximately 90 amino acids andencodes a central sequence of 8 aromatic and positively charged residues.33 This association of AuNPs with RNA or RNA relatedprotein machinery emerged again when we examined enrichment of biologicalpathways (Figure 6b) and gene ontology (GO)based functional enrichment (Supporting Information, Figure S7). Pathways involving ribosome, spliceosome, and geneexpression were some of the most enriched pathways along with GO termssuch as RNA binding and structural constituent of ribosome. The positivecharge of RRM_1 and the “plastic” nature of the domainmay explain the enrichment of the domain in the NP-protein complex.Similar structural flexibility capabilities that allow RNA relatedmachinery to interact with RNA might be at play for interaction withAuNPs, and hence, we see ribosome and spliceosome as one of the mostenriched pathways that the protein-NP complex proteins belong to.This association of AuNPs with the RNA proteins might also explainwhy 20 nm AuNPs inhibited proliferation of cancer cells and reversedEMT by down-regulating transcription and secretion of multiple proteins.20,34 Also enriched on the NP surface were proteins involved in proteinfolding and cytoskeleton-related processes and functions.


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)

Analysis ofevolutionarily conserved functional domains (a) andbiological pathways (b) enriched in proteins detected at differenttime points revealed that AuNPs have a high affinity to mRNA relatedprotein machinery. Protein domains from Pfam database and pathwaysfrom databases KEGG (hsa*), Panther (P0*), and Reactome (REACT_*)that are significantly enriched among one or more lists are shown.Color of matrices indicates Bonferroni corrected P-values (P ≤ 0.05). Green indicates higherenrichment, red indicates lower enrichment, and gray color indicatesthat a given term did not reach statistical significance among theproteins in the list. Pathway enrichment analyses were performed usingDAVID v 6.1 using default settings. Human proteome was defined asthe background. (M = min, H = hours).
© Copyright Policy
Related In: Results  -  Collection

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

fig6: Analysis ofevolutionarily conserved functional domains (a) andbiological pathways (b) enriched in proteins detected at differenttime points revealed that AuNPs have a high affinity to mRNA relatedprotein machinery. Protein domains from Pfam database and pathwaysfrom databases KEGG (hsa*), Panther (P0*), and Reactome (REACT_*)that are significantly enriched among one or more lists are shown.Color of matrices indicates Bonferroni corrected P-values (P ≤ 0.05). Green indicates higherenrichment, red indicates lower enrichment, and gray color indicatesthat a given term did not reach statistical significance among theproteins in the list. Pathway enrichment analyses were performed usingDAVID v 6.1 using default settings. Human proteome was defined asthe background. (M = min, H = hours).
Mentions: We next asked whether the proteins that were attached tothe NPsshared conserved domains32 which couldshed light on why some proteins adsorb onto the NP surface while othersdo not. Of the many domains that were enriched, from the proteinsbound to the NP surface RRM_1, a RNA recognition motif, was enrichedin proteins from all time point complexes (Figure 6a). The RRM_1 a motif is approximately 90 amino acids andencodes a central sequence of 8 aromatic and positively charged residues.33 This association of AuNPs with RNA or RNA relatedprotein machinery emerged again when we examined enrichment of biologicalpathways (Figure 6b) and gene ontology (GO)based functional enrichment (Supporting Information, Figure S7). Pathways involving ribosome, spliceosome, and geneexpression were some of the most enriched pathways along with GO termssuch as RNA binding and structural constituent of ribosome. The positivecharge of RRM_1 and the “plastic” nature of the domainmay explain the enrichment of the domain in the NP-protein complex.Similar structural flexibility capabilities that allow RNA relatedmachinery to interact with RNA might be at play for interaction withAuNPs, and hence, we see ribosome and spliceosome as one of the mostenriched pathways that the protein-NP complex proteins belong to.This association of AuNPs with the RNA proteins might also explainwhy 20 nm AuNPs inhibited proliferation of cancer cells and reversedEMT by down-regulating transcription and secretion of multiple proteins.20,34 Also enriched on the NP surface were proteins involved in proteinfolding and cytoskeleton-related processes and functions.

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