전체메뉴
검색
Article Search

JMB Journal of Microbiolog and Biotechnology

QR Code QR Code

Note

References

  1. Prusiner SB. 1998. Prions. Proc. Natl. Acad. Sci. USA 95: 13363-13383.
    Pubmed PMC CrossRef
  2. Prusiner SB, McKinley MP, Bowman KA, Bolton DC, Bendheim PE, Groth DF, et al. 1983. Scrapie prions aggregate to form amyloid-like birefringent rods. Cell 35: 349-358.
    Pubmed CrossRef
  3. Aguzzi A, Lakkaraju AKK, Frontzek K. 2018. Toward therapy of human prion diseases. Annu. Rev. Pharmacol. Toxicol. 58: 331-351.
    Pubmed CrossRef
  4. Solassol J, Crozet C, Perrier V, Leclaire J, Beranger F, Caminade A-M, et al. 2004. Cationic phosphorus-containing dendrimers reduce prion replication both in cell culture and in mice infected with scrapie. J. Gen. Virol. 85: 1791-1799.
    Pubmed CrossRef
  5. Cordes H, Boas U, Olsen P, Heegaard PMH. 2007. Guanidino- and urea-modified dendrimers as potent solubilizers of misfolded prion protein aggregates under non-cytotoxic conditions: dependence on dendrimer generation and surface charge. Biomacromolecules 8: 3578-3583.
    Pubmed CrossRef
  6. Supattapone S, Nguyen H-OB, Cohen FE, Prusiner SB, Scott MR. 1999. Elimination of prions by branched polyamines and implications for therapeutics. Proc. Natl. Acad. Sci. USA 96: 14529-14534.
    Pubmed PMC CrossRef
  7. Supattapone S, Wille H, Uyechi L, Safar J, Tremblay P, Szoka FC, et al. 2001. Branched polyamines cure prioninfected neuroblastoma cells. J. Virol. 75: 3453-3461.
    Pubmed PMC CrossRef
  8. Lim Y-b, Mays CE, Kim Y, Titlow WB, Ryou C. 2010. The inhibition of prions through blocking prion conversion by permanently charged branched polyamines of low cytotoxicity. Biomaterials 31: 2025-2033.
    Pubmed CrossRef
  9. Jackson KS, Yeom J, Han Y, Bae Y, Ryou C. 2013. Preference toward a polylysine enantiomer in inhibiting prions. Amino Acids 44: 993-1000.
    Pubmed CrossRef
  10. Ryou C, Titlow WB, Mays CE, Bae Y, Kim S. 2011. The suppression of prion propagation using poly-l-lysine by targeting plasminogen that stimulates prion protein conversion. Biomaterials 32: 3141-3149.
    Pubmed PMC CrossRef
  11. Titlow WB, Waqas M, Lee J, Cho JY, Lee SY, Kim DH, et al. 2016. Effect of polylysine on scrapie prion protein propagation in spleen during asymptomatic stage of experimental prion disease in mice. J. Microbiol. Biotechnol. 26: 1657-1660.
    Pubmed CrossRef
  12. Waqas M, Lee H-M, Kim J, Telling G, Kim J-K, Kim D-H, et al. 2017. Effect of poly-L-arginine in inhibiting scrapie prion protein of cultured cells. Mol. Cell. Biochem. 428: 57-66.
    Pubmed PMC CrossRef
  13. Waqas M, Jeong W-j, Lee Y-J, Kim D-H, Ryou C, Lim Y-b. 2017. pH-dependent in-cell self-assembly of peptide inhibitors increases the anti-prion activity while decreasing the cytotoxicity. Biomacromolecules 18: 943-950.
    Pubmed CrossRef
  14. Xu Z, Adrover M, Pastore A, Prigent S, Mouthon F, Comoy E, et al. 2011. Mechanistic insights into cellular alteration of prion by poly-D-lysine: the role of H2H3 domain. FASEB J. 25: 3426-3435.
    Pubmed CrossRef
  15. Bond VC, Wold B. 1987. Poly-L-ornithine-mediated transformation of mammalian cells. Mol. Cell. Biol. 7: 2286-2293.
    Pubmed PMC CrossRef
  16. Ge H, Tan L, Wu P, Yin Y, Liu X, Meng H, et al. 2015. Poly-L-ornithine promotes preferred differentiation of neural stem/progenitor cells via ERK signalling pathway. Sci. Rep. 5: 15535.
    Pubmed PMC CrossRef
  17. Lee ES, Na K, Bae YH. 2005. Super pH-sensitive multifunctional polymeric micelle. Nano Lett. 5: 325-329.
    Pubmed CrossRef
  18. Scott MRD, Butler DA, Bredesen DE, Walchli M, Hsiao KK, Prusiner SB. 1988. Prion protein gene expression in cultured cells. Prot. Eng. 2: 69-76.
    Pubmed CrossRef
  19. Arnold JE, Tipler C, Laszlo L, Hope J, Landon M, Mayer RJ. 1995. The abnormal isoform of the prion protein accumulates in late-endosome-like organelles in scrapie-infected mouse brain. J. Pathol. 176: 403-411.
    Pubmed CrossRef

Related articles in JMB

More Related Articles

Article

Note

J. Microbiol. Biotechnol. 2018; 28(12): 2141-2144

Published online December 28, 2018 https://doi.org/10.4014/jmb.1807.07045

Copyright © The Korean Society for Microbiology and Biotechnology.

Decrease of protease-resistant PrPSc level in ScN2a cells by polyornithine and polyhistidine

Muhammad Waqas 1, Huyen Trang Trinh 1, Sungeun Lee 1, Dae-hwan Kim 1, 2, Sang Yeol Lee 3, Kevin K Choe 1 and Chongsuk Ryou 1*

1Department of Pharmacy and Institute of Pharmaceutical Science & Technology, Hanyang University, 2School of Undergraduate Studies, Daegu Gyeongbuk Institute of Science and Technology (DGIST), 3Department of Life Science, Gachon University, Seongnam, Gyeonggi-do 13120, Republic of Korea

Received: July 24, 2018; Accepted: October 22, 2018

Abstract

Based on previous studies reporting anti-prion activity of poly-L-lysine and poly-L-arginine, cationic poly-L-ornithine (PLO) and poly-L-histidine (PLH), anionic poly-L-glutamic acid (PLE) and uncharged poly-L-threonine (PLT) were investigated in cultured cells chronically infected by prions to determine anti-prion efficacy. While PLE and PLT did not alter the level of PrPSc, PLO and PLH exhibited potent PrPSc inhibition in ScN2a cells. These results suggest that anti-prion activity of poly-basic amino acids is correlated with cationicity of their functional groups. Comparison of anti-prion activity of PLO and PLH proposes that anti-prion activity of poly-basic amino acids is associated with the acidic cellular compartments.

Keywords: Prion, polyornithine, polyhistidine, cationic amino acid polymer

Body

Prion diseases are fatal, progressive neurodegenerative conditions in humans and animals [1]. The normal cellular form of prion protein (PrPC) is conformationally changed to the pathogenic isoform of prion protein (PrPSc), which is the sole component of prion agents [1]. Accumulation of PrPSc in the brain results in neuronal damage and subsequent cell death, leading to degeneration of the central nervous system [2].

Unfortunately, there is no treatment available for prion diseases [3]. Among a number of attempts to discover effective anti-prion agents, a group of studies reported that cationic compounds exhibit potent activity in inhibiting prions [4-14]. In particular, our group demonstrated that poly-L-lysine (PLK) suppresses PrPSc propagation in various systems, including the cell-free, cultured cell, and mouse models of prion diseases [10, 11]. In the following study, we showed that poly-D-lysine, an enantiomer of PLK, retains greater anti-prion potency as well as cytotoxicity than PLK [9, 14]. Similarly, we also found poly-L-arginine (PLR) inhibits PrPSc more efficiently in cultured cells in which prions of different origins propagate [12]. Furthermore, nanostructures made of oligo-L-arginine showed comparable anti-prion activity to PLR, while reducing the cytotoxicity level [13]

Poly-L-ornithine (PLO) is a cationic polymer composed of L-ornithine, a metabolic intermediate of L-arginine. Like PLK and PLR, PLO has been used as a DNA transfection agent into mammalian cells and a medium to attach cells onto the culture containers [15, 16]. Poly-L-histidine (PLH) is comprised of L-histidine, which is responsible for most of the buffering competence of proteins in the physiological pH array due to its pKa value. By the same reason, PLH is a pH-responsive polymeric carrier and has been used as an endosomal pH targeting agent [17].

In this study, we investigated the anti-prion efficacy of constitutive and conditional cationic amino acid polymers, PLO and PLH, respectively, in cultured cells with permanent prion infection, together with PLR previously shown to exhibit potent anti-prion activity [12]. To confirm the relationship between anti-prion activity and the cationic property of amino acid polymers, poly-L-glutamic acid (PLE), an anionic poly-amino acid, and poly-L-threonine (PLT), a polar but electrically uncharged poly-amino acid, were also examined for anti-prion efficacy.

Poly-amino acids used in this study, PLO10, PLH10, PLR10, PLE22.5, and PLT22.5 (Figs. 1A and 2A), were purchased from Sigma-Aldrich (USA). The average molecular mass (kDa) of these polymers was shown as suffix numbers in their names. To measure the anti-prion activity of poly-amino acids, the culture of ScN2a cells [18], incubation of cells with amino acid polymers, and assays to examine the levels of PrPSc, a biochemical marker for prion replication, were performed as described previously [10, 12]. Initially, 4 × 106 cells were seeded in culture dishes (100 mm) and cultured in Dulbecco’s Modified Eagle’s Medium (DMEM) containing 10% fetal bovine serum, 1%penicillin-streptomycin, and 1% Glutamax. Cell culture reagents were purchased from Invitrogen (Carlsbad, USA). Cells were incubated at 37°C, 5% CO2 and saturated humidity. As seeded cells attached on the surface of culture dishes, various concentrations of amino acid polymers were added to the culture media. Incubation lasted for six days and on the fourth day media were replaced with the fresh culture media containing polymers. Then, cell lysate was prepared in 1 ml cell lysis buffer (20 mM Tris, pH 8.0; 0.5% Nonidet P-40; 0.5% sodium deoxycholate; 150mM NaCl). Cell lysate (~30 μg of protein) was analyzed to measure the levels of total PrP and βIII tubulin loading controls by western blotting using anti-PrP antibody 6D11 (Covance, Dedham, USA) and anti- βIII tubulin antibody (R&D System, Minneapolis, USA). For proteinase K (PK)-resistant PrPSc preparation, cell lysate (2 mg of protein) was incubated with PK (20 μg/ml) for 1 h at 37°C and centrifuged for 1 h at 16,000 ×g at 4°C. PrPSc in the pellet was subjected to analysis. Protein bands in western blots were visualized using ECL Prime Detection Reagents (Amersham, GE Healthcare, Piscataway, USA) and detected by G:Box Chemi XR5 system (Syngene, Cambridge, UK). The viability of ScN2a cells incubated with amino acid polymers was measured using the 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) assay protocol described previously [10, 12]. Briefly, ScN2a cells in a 24-well culture plate were seeded and incubated with amino acid polymers in the same manner as described earlier. Then, cells were incubated with DMEM containing 0.5 mg/ml MTT (Sigma-Aldrich, USA) for an additional 3 h. MTT formazan products were extracted with 0.05 N HCl-isopropanol and quantified through colorimetric readouts at 570 nm using an Infinite M200Pro Multimode Reader (Tecan, Männedorf, Switzerland).

Figure 1. Anti-prion efficacy of PLO10, PLH10, PLR10 in ScN2a cells. (A) Structures of PLO10, PLH10, PLR10. The numbers within the parentheses indicate repeated unit counts. (B) Western blots of PK-resistant PrPSc in ScN2a cells incubated with 0-400 nM PLO10, PLH10, and PLR10. (C) Western blots of βIII tubulin in ScN2a cells incubated with 0-400 nM PLO10, PLH10, and PLR10. (D) Survival of ScN2a cells incubated with 0-1,000 nM PLO10, PLH10, and PLR10. The shaded box represents the concentration range used for efficacy tests in Panel B. Survival rates at each data point were obtained from the average of triplicate assays and the error bars indicate the standard deviation. Western blotting and cytotoxicity assays were confirmed by at least more than three independent experiments.

Figure 2. Anti-prion efficacy of PLE22.5 and PLT22.5 in ScN2a cells. (A) Structures of PLE22.5 and PLT22.5. The numbers within the parentheses indicate repeated unit counts. (B) Western blots of PK-resistant PrPSc in ScN2a cells incubated with 0-400 nM PLE22.5 and PLT22.5. (C) Western blots of βIII tubulin in ScN2a cells incubated with 0-400 nM PLE22.5 and PLT22.5. (D) Survival of ScN2a cells incubated with 0-1,000 nM PLE22.5 and PLT22.5. The shaded box represents the concentration range used for efficacy tests in Panel B. Survival rates at each data point were obtained from the average of triplicate assays and the error bars indicate the standard deviation. Western blotting and cytotoxicity assays were confirmed by at least more than three independent experiments.

To measure anti-prion efficacy of PLO and PLH in comparison to PLR, ScN2a cells were incubated with various concentrations of PLO10, PLH10, and PLR10 and the level of PK-resistant PrPSc was monitored. Western blot analysis showed that both PLO10 and PLH10 effectively decreased the level of PrPSc in ScN2a cells in a concentration-dependent manner (Fig. 1B). The level of loading control, βIII tubulin, remained constant (Fig. 1C). The dose responsiveness to inhibit PrPSc propagation by the low concentrations of PLO10 and PLH10 was less sensitive than by the corresponding concentrations of PLR10. This indicates that efficiency of PrPSc inhibition varies for different cationic poly-amino acids, presumably due to the functional group of each amino acid. This suggests that the guanidinium groups in PLR are more potent than the amine groups in PLO to inhibit PrPSc propagation. Unlike constitutively cationic PLO and PLR, PLH conditionally becomes cationic under acidic local environment owing to protonation of the imidazole ring of histidine, which occurs at pH below its pKa (~6.0). Hence, anti-prion activity exerted by PLH suggests that inhibition of PrPSc propagation by cationic poly-amino acids is facilitated in the acidic subcellular compartments, presumably within the endosomes or lysosomes known to be the subcellular organelles where PrPSc is converted from PrPC and accumulated as aggregates, respectively [19]. The results of cytotoxicity tests for PLO10, PLH10, and PLR10 showed that these amino acid polymers were, overall, not toxic (Fig. 1D). The concentrations of PLO10, PLH10, and PLR10, displaying effective anti-prion activity, were at non-toxic concentrations. This suggests that anti-prion activity achieved by PLO10, PLH10, and PLR10 was attributed to inhibitory activity of the polymers, but not to the death of prion-infected cells caused by their toxic effect.

To authenticate the correlation of cationic property of PLO, PLR, and PLH to anti-prion activity, anionic poly-amino acids PLE22.5 and electrically uncharged poly-amino acids PLT22.5 were examined to determine whether they affect the level of PrPSc in ScN2a cells. Incubation of cells with PLE22.5 and PLT22.5 did not change the level of PK-resistant PrPSc (Fig. 2B). The level of loading control, βIII tubulin, and cell survival were not affected by PLE22.5 and PLT22.5 (Figs. 2B and 2C). These results indicate that amino acid polymers with negative or no charges are not able to inhibit PrPSc propagation.

In conclusion, anti-prion activity exhibited by PLO, PLR and PLH is attributed to the cationicity of poly-amino acids. It appears that inhibition of PrPSc propagation by basic amino acid polymers is facilitated in the acidic subcellular organelles.

Acknowledgment

This work was supported by the research fund of Hanyang University (HY-2014-P)

Conflict of Interest


The authors have no financial conflicts of interest to declare.

Fig 1.

Figure 1.Anti-prion efficacy of PLO10, PLH10, PLR10 in ScN2a cells. (A) Structures of PLO10, PLH10, PLR10. The numbers within the parentheses indicate repeated unit counts. (B) Western blots of PK-resistant PrPSc in ScN2a cells incubated with 0-400 nM PLO10, PLH10, and PLR10. (C) Western blots of βIII tubulin in ScN2a cells incubated with 0-400 nM PLO10, PLH10, and PLR10. (D) Survival of ScN2a cells incubated with 0-1,000 nM PLO10, PLH10, and PLR10. The shaded box represents the concentration range used for efficacy tests in Panel B. Survival rates at each data point were obtained from the average of triplicate assays and the error bars indicate the standard deviation. Western blotting and cytotoxicity assays were confirmed by at least more than three independent experiments.
Journal of Microbiology and Biotechnology 2018; 28: 2141-2144https://doi.org/10.4014/jmb.1807.07045

Fig 2.

Figure 2.Anti-prion efficacy of PLE22.5 and PLT22.5 in ScN2a cells. (A) Structures of PLE22.5 and PLT22.5. The numbers within the parentheses indicate repeated unit counts. (B) Western blots of PK-resistant PrPSc in ScN2a cells incubated with 0-400 nM PLE22.5 and PLT22.5. (C) Western blots of βIII tubulin in ScN2a cells incubated with 0-400 nM PLE22.5 and PLT22.5. (D) Survival of ScN2a cells incubated with 0-1,000 nM PLE22.5 and PLT22.5. The shaded box represents the concentration range used for efficacy tests in Panel B. Survival rates at each data point were obtained from the average of triplicate assays and the error bars indicate the standard deviation. Western blotting and cytotoxicity assays were confirmed by at least more than three independent experiments.
Journal of Microbiology and Biotechnology 2018; 28: 2141-2144https://doi.org/10.4014/jmb.1807.07045

References

  1. Prusiner SB. 1998. Prions. Proc. Natl. Acad. Sci. USA 95: 13363-13383.
    Pubmed KoreaMed CrossRef
  2. Prusiner SB, McKinley MP, Bowman KA, Bolton DC, Bendheim PE, Groth DF, et al. 1983. Scrapie prions aggregate to form amyloid-like birefringent rods. Cell 35: 349-358.
    Pubmed CrossRef
  3. Aguzzi A, Lakkaraju AKK, Frontzek K. 2018. Toward therapy of human prion diseases. Annu. Rev. Pharmacol. Toxicol. 58: 331-351.
    Pubmed CrossRef
  4. Solassol J, Crozet C, Perrier V, Leclaire J, Beranger F, Caminade A-M, et al. 2004. Cationic phosphorus-containing dendrimers reduce prion replication both in cell culture and in mice infected with scrapie. J. Gen. Virol. 85: 1791-1799.
    Pubmed CrossRef
  5. Cordes H, Boas U, Olsen P, Heegaard PMH. 2007. Guanidino- and urea-modified dendrimers as potent solubilizers of misfolded prion protein aggregates under non-cytotoxic conditions: dependence on dendrimer generation and surface charge. Biomacromolecules 8: 3578-3583.
    Pubmed CrossRef
  6. Supattapone S, Nguyen H-OB, Cohen FE, Prusiner SB, Scott MR. 1999. Elimination of prions by branched polyamines and implications for therapeutics. Proc. Natl. Acad. Sci. USA 96: 14529-14534.
    Pubmed KoreaMed CrossRef
  7. Supattapone S, Wille H, Uyechi L, Safar J, Tremblay P, Szoka FC, et al. 2001. Branched polyamines cure prioninfected neuroblastoma cells. J. Virol. 75: 3453-3461.
    Pubmed KoreaMed CrossRef
  8. Lim Y-b, Mays CE, Kim Y, Titlow WB, Ryou C. 2010. The inhibition of prions through blocking prion conversion by permanently charged branched polyamines of low cytotoxicity. Biomaterials 31: 2025-2033.
    Pubmed CrossRef
  9. Jackson KS, Yeom J, Han Y, Bae Y, Ryou C. 2013. Preference toward a polylysine enantiomer in inhibiting prions. Amino Acids 44: 993-1000.
    Pubmed CrossRef
  10. Ryou C, Titlow WB, Mays CE, Bae Y, Kim S. 2011. The suppression of prion propagation using poly-l-lysine by targeting plasminogen that stimulates prion protein conversion. Biomaterials 32: 3141-3149.
    Pubmed KoreaMed CrossRef
  11. Titlow WB, Waqas M, Lee J, Cho JY, Lee SY, Kim DH, et al. 2016. Effect of polylysine on scrapie prion protein propagation in spleen during asymptomatic stage of experimental prion disease in mice. J. Microbiol. Biotechnol. 26: 1657-1660.
    Pubmed CrossRef
  12. Waqas M, Lee H-M, Kim J, Telling G, Kim J-K, Kim D-H, et al. 2017. Effect of poly-L-arginine in inhibiting scrapie prion protein of cultured cells. Mol. Cell. Biochem. 428: 57-66.
    Pubmed KoreaMed CrossRef
  13. Waqas M, Jeong W-j, Lee Y-J, Kim D-H, Ryou C, Lim Y-b. 2017. pH-dependent in-cell self-assembly of peptide inhibitors increases the anti-prion activity while decreasing the cytotoxicity. Biomacromolecules 18: 943-950.
    Pubmed CrossRef
  14. Xu Z, Adrover M, Pastore A, Prigent S, Mouthon F, Comoy E, et al. 2011. Mechanistic insights into cellular alteration of prion by poly-D-lysine: the role of H2H3 domain. FASEB J. 25: 3426-3435.
    Pubmed CrossRef
  15. Bond VC, Wold B. 1987. Poly-L-ornithine-mediated transformation of mammalian cells. Mol. Cell. Biol. 7: 2286-2293.
    Pubmed KoreaMed CrossRef
  16. Ge H, Tan L, Wu P, Yin Y, Liu X, Meng H, et al. 2015. Poly-L-ornithine promotes preferred differentiation of neural stem/progenitor cells via ERK signalling pathway. Sci. Rep. 5: 15535.
    Pubmed KoreaMed CrossRef
  17. Lee ES, Na K, Bae YH. 2005. Super pH-sensitive multifunctional polymeric micelle. Nano Lett. 5: 325-329.
    Pubmed CrossRef
  18. Scott MRD, Butler DA, Bredesen DE, Walchli M, Hsiao KK, Prusiner SB. 1988. Prion protein gene expression in cultured cells. Prot. Eng. 2: 69-76.
    Pubmed CrossRef
  19. Arnold JE, Tipler C, Laszlo L, Hope J, Landon M, Mayer RJ. 1995. The abnormal isoform of the prion protein accumulates in late-endosome-like organelles in scrapie-infected mouse brain. J. Pathol. 176: 403-411.
    Pubmed CrossRef