Cytotoxicity of V30M and L55P Transthyretin Variants on Human Cells
Abstract
Transthyretin (TTR) is one of the thyroxine transport protein in the plasma and cerebrospinal fluid. It is a 55-kDa homotetrameric protein. The mutation of TTR gene that leads to a single amino acid change of TTR protein will result in the TTR variant, which is the major cause of TTR amyloidosis. TTR mis-folding into toxic amyloid fibril found deposited and resulting in dysfunction of the nervous system. Among the identified TTR variants, V30M (methionine for valine substitution at position 30) is the most common type; whereas, L55P (proline for leucine substitution at position 55) is the most aggressive type. To evaluate amyloidogenicity of TTR variants, cytotoxicity assay is a primary assessment; however, the sensitivity and specificity of cell to TTR amyloid are the effectiveness factors of the assay. The objective of this research is to comparatively study on the responsiveness of human cell types to the toxicity of TTR amyloid in order to identify more cell that is sensitive and appropriate for using as a tool in the study of TTR amyloidosis. By using recombinant V30M and L55P as tools, it revealed that L55P was induced by acidic condition to form a soluble aggregate faster than V30M, and it was more toxic to the studied cells including fibroblasts (F‑N and F-DS), neuroblast (LAN-5) and lymphoblast than V30M. In addition, the comparatively results showed that F-DS cells were the most sensitive to the toxicity of soluble aggregated TTRs. Keywords : cytotoxicity ; fibroblast ; lymphoblast ; neuroblast ; recombinant proteinReferences
Barreiros, A.P., Otto, G., Kahlen, B., Teufel, A., & Galle, P.R. (2015). Familial amyloidosis : Great progress for
an orphan disease. Journal of Hepatology, 62(2), 483-485.
Bradford, M.M. (1976). A rapid and sensitive method for the quantitation of microgram quantities of protein
utilizing the principle of protein-dye binding. Analytical Biochemistry, 72(1-2), 248-254.
Cascella, R., Conti, S., Mannini, B., Li, X., Buxbaum, J., Tiribilli, B., Chiti, F., & Cecchi, C. (2013). Transthyretin
suppresses the toxicity of oligomers formed by misfolded proteins in vitro. Biochimica et Biophysica Acta,
1832(12), 2302-2314.
Cereghino, J.L., & Cregg, J.M. (2000). Heterologous protein expression in the methylotrophic yeast Pichia
pastoris. FEMS Microbiology Reviews, 24(1), 45-66.
Christmanson, L., Betsholtz, C., Gustavsson, A., Johansson, B., Sletten, K., & Westermark, P. (1991).
The transthyretin cDNA sequence is normal in transthyretin-derived senile systemic amyloidosis.
FEBS Letters, 281(1-2), 177-180.
Coelho, T., Adams, D., Silva, A., Lozeron, P., Hawkins, P.N., Mant, T., Perez, J., Chiesa, J., Warrington, S., Tranter,
E., Munisamy, M., Falzone, R. Harrop, J., Cehelsky, J., Bettencourt, B.R., Geissler, M., Butler, J.S.,
Sehgal, A., Meyers, R.E., Chen, Q., Borland, T., Hutabarat, R.M., Clausen, V.A., Alvarez, R., Fitzgerald, K.,
Gamba-Vitalo, C., Nochur, S.V., Vaishnaw, A.K., Sah, D.W., Gollob, J..A., & Suhr, O.B. (2013). Safety and
efficacy of RNAi therapy for transthyretin amyloidosis. The New England Journal of Medicine, 369(9), 819-829.
Daly, R., & Hearn, M.T. (2005). Expression of heterologous proteins in Pichia pastoris: a useful experimental tool in
protein engineering and production. Journal of Molecular Recognition, 18(2), 119-138.
Dasari, A.K.R., Hughes, R.M., Wi, S., Hung, I., Gan, Z., Kelly, J.W. & Lim, K.H. (2019). Transthyretin aggregation
pathway toward the formation of distinct cytotoxic oligomers. Scientific Reports, 9, 33-43.
Dasari, A.K.R., Hung, I., Gan, Z., & Lim, K.H. (2020). Two distinct aggregation pathways in transthyretin misfolding
and amyloid formation. Biochimica et Biophysica Acta (BBA) - Proteins and Proteomics, 1867(3),
344-349.
Duan, W., Richardson, S.J., Köhrle, J., Chang, L., Southwell, B.R., Harms, P.J., Brack, C.M., Pettersson, T.M., &
Schreiber. G. (1995). Binding of thyroxine to pig transthyretin, its cDNA structure, and other properties.
European Journal of Biochemistry, 230(3), 977-986.
Ferreira, N., Saraiva, M.J., & Almeida, M.R. (2019). Uncovering the neuroprotective mechanisms of curcumin on
transthyretin amyloidosis. International Journal of Molecular Sciences, 20(6), 1287.
Garcia-Alloza, M., Borrelli L.A., Rozkalne, A., Hyman B.T., & Bacskai, B.J. (2007). Curcumin labels amyloid
pathology in vivo, disrupts existing plaques, and partially restores distorted neurites in an Alzheimer
mouse model. Journal of Neurochemistry, 102(4), 1095-1104.
Holmgren, G., Ericzon, B.G., Groth, C.G., Steen, L., Suhr, O., Andersen, O., Wallin, B.G., Seymour, A., Richardson,
S., Hawkins, P.N., & Pepys, M.B. (1993). Clinical improvement and amyloid regression after liver
transplantation in hereditary transthyretin amyloidosis. The Lancet, 341(8853), 1113-1116.
Hou, X., Richardson, S.J., Aguilar, M-I., & Small, D.H. (2005). Binding of amyloidogenic transthyretin to the plasma
membrane alters membrane fluidity and induces neurotoxicity. Biochemistry, 44(34), 11618-11627.
Hund, E., Linke, R.P., Willig, F. & Gra, A. (2001). Transthyretin-associated neuropathic amyloidosis. Pathogenesis
and treatment. Neurology, 56(4), 431-435.
Jacobson, D.R., & Buxaum, J.N. (1991). Genetic aspects of amyloidosis. Advances in Human Genetics, 20,
69-123.
Kaewmeechai, S., Poodproh, R., & Prapunpoj P. (2019). Transthyretin variant in Thai people is likely to associate
with pathogenesis. Rajamangala University of Technology Srivijaya Research Journal, 11(3), 387-401.
Kawas, C.H. (2006). Medications and diet protective factors for AD?. Alzheimer Disease & Associated Disorders,
20(3 suppl 2), S89–S96.
Lashuel, H.A., Lai, Z., & Kelly, J.W. (1998). Characterization of the transthyretin acid denaturation pathways by
analytical ultracentrifugation: implications for wild-type, V30M, and L55P amyloid fibril formation.
Biochemistry Journal, 37(51), 17851-17864.
Park, G.Y., Jamerlan, A., Shim, K.H., & An, S.S.A. (2019). Diagnostic and treatment approaches involving
Transthyretin in amyloidogenic diseases. International Journal of Molecular Sciences, 20(12), 2982-2998.
Peterson, S.A., Klabunde, T., Lashuel, H.A., Purkey, H., Sacchettini, J.C., & Kelly, J.W. (1998). Inhibiting
transthyretin conformational changes that lead to amyloid fibril formation. Proceedings of the National
Academy of Sciences of the United States of America, 95(22), 12956-12960.
Prapunpoj, P., Richardson, S.J., & Schreiber, G. (2002). Crocodile transthyretin: structure, function, and evolution.
American Journal of Physiology-Regulatory, Integrative and Comparative Physiology, 283(4),
R885–R896.
Prapunpoj, P., Yamauchi, K., Nishiyama, N., Richardson, S.J., & Schreiber, G. (2000). Evolution of structure,
ontogeny of gene expression, and function of Xenopus laevis transthyretin. American Journal of
Physiology-Regulatory, Integrative and Comparative Physiology, 279, R2026-R204.
Reixach, N., Deechongkit, S., Jiang, X., Kelly, J.W., & Buxbaum, J.N. (2004). Tissue damage in the amyloidoses:
Transthyretin monomers and nonnative oligomers are the major cytotoxic species in tissue culture.
Proceedings of the National Academy of Sciences of the United States of America, 101(9), 2817-2822.
Richardson, S.J. (2007). Cell and molecular biology of transthyretin and thyroid hormones. International Review of
Cytology, 258, 137-193.
Ruberg, F.L. & Berk, J.L. (2012). Transthyretin (TTR) cardiac amyloidosis. Circulation, 126(10), 1286-1300.
Saelices, L., Johnson, L.M., Liang, W.Y., Sawaya, M.R., Cascio, Duilio, Ruchala, P., Whitelegge, J., Jiang, L., Riek,
R., & Eisenberg, D.S. (2015). Uncovering the mechanism of aggregation of human transthyretin. Journal
of Biological Chemistry, 290(48), 28932-28943.
Sorgjerd, K., Klingstedt, T., Lindgren, M., Kagedal, K., & Hammarstrom, P. (2008). Prefibrillar transthyretin
oligomers and cold stored native tetrameric transthyretin are cytotoxic in cell culture. Biochemical and
Biophysical Research Communications, 377(4), 1072-1078.
Sousa, M.M., & Saraiva, M. (2003). Neurodegeneration in familial amyoloid polyneuropathy: from pathology to
molecular signaling. Progress in Neurobiology, 71(5), 385-400.
Tan, S.Y., Pepys, M.B., & Hawkins, P.N. (1995). Treatment of amyloidosis. American Journal of Kidney Diseases,
26(2), 267-285.
Tola, A.J, Leelawatwattana, L., & Prapunpoj, P. (2019). The catalytic kinetics of chicken transthyretin towards
human A1-42. Comparative Biochemistry and Physiology Part C: Toxicology & Pharmacology, 226,
doi: 10.1016/j.cbpc.2019.108610.
Vogl, T., & Glieder, A. (2013). Regulation of Pichia pastoris promoters and its consequences for protein
production. New Biotechnology, 30(4), 385-404.
Wang, X., Cattaneo, F., Ryno, L., Hulleman, J., Reixach, N., & Buxbaum, J. (2014). The systemic amyloid precursor
transthyretin (TTR) behaves as a neuronal stress protein regulated by HSF1 in SH-SY5Y human
neuroblastoma cells and APP23 Alzheimer’s disease model mice. The Journal of Neuroscience, 34(21),
7253-7265.
Xue, Q., Zheng, Q.C., Zhang, J.L., Cui, Y.L. Chu, W.T., & Zhang, H.X. (2014). Mutation and low pH effect on the
stability as well as unfolding kinetics of transthyretin dimer. Biophysical Chemistry, 189, 8-15.
Yang, M., Lei, M., & Huo, S. (2003). Why is Leu55→Pro55 transthyretin variant the most amyloidgenic; Insights
from molecular dynamics simulations of transthyretin monomers. Protein Science, 12(6), 1222-1231.
Yiannopoulou, K.G., & Papageorgiou, S.G. (2013). Current and future treatments for Alzheimer’s disease.
Therapeutic Advances in Neurological Disorders, 6(1), 19-33.
an orphan disease. Journal of Hepatology, 62(2), 483-485.
Bradford, M.M. (1976). A rapid and sensitive method for the quantitation of microgram quantities of protein
utilizing the principle of protein-dye binding. Analytical Biochemistry, 72(1-2), 248-254.
Cascella, R., Conti, S., Mannini, B., Li, X., Buxbaum, J., Tiribilli, B., Chiti, F., & Cecchi, C. (2013). Transthyretin
suppresses the toxicity of oligomers formed by misfolded proteins in vitro. Biochimica et Biophysica Acta,
1832(12), 2302-2314.
Cereghino, J.L., & Cregg, J.M. (2000). Heterologous protein expression in the methylotrophic yeast Pichia
pastoris. FEMS Microbiology Reviews, 24(1), 45-66.
Christmanson, L., Betsholtz, C., Gustavsson, A., Johansson, B., Sletten, K., & Westermark, P. (1991).
The transthyretin cDNA sequence is normal in transthyretin-derived senile systemic amyloidosis.
FEBS Letters, 281(1-2), 177-180.
Coelho, T., Adams, D., Silva, A., Lozeron, P., Hawkins, P.N., Mant, T., Perez, J., Chiesa, J., Warrington, S., Tranter,
E., Munisamy, M., Falzone, R. Harrop, J., Cehelsky, J., Bettencourt, B.R., Geissler, M., Butler, J.S.,
Sehgal, A., Meyers, R.E., Chen, Q., Borland, T., Hutabarat, R.M., Clausen, V.A., Alvarez, R., Fitzgerald, K.,
Gamba-Vitalo, C., Nochur, S.V., Vaishnaw, A.K., Sah, D.W., Gollob, J..A., & Suhr, O.B. (2013). Safety and
efficacy of RNAi therapy for transthyretin amyloidosis. The New England Journal of Medicine, 369(9), 819-829.
Daly, R., & Hearn, M.T. (2005). Expression of heterologous proteins in Pichia pastoris: a useful experimental tool in
protein engineering and production. Journal of Molecular Recognition, 18(2), 119-138.
Dasari, A.K.R., Hughes, R.M., Wi, S., Hung, I., Gan, Z., Kelly, J.W. & Lim, K.H. (2019). Transthyretin aggregation
pathway toward the formation of distinct cytotoxic oligomers. Scientific Reports, 9, 33-43.
Dasari, A.K.R., Hung, I., Gan, Z., & Lim, K.H. (2020). Two distinct aggregation pathways in transthyretin misfolding
and amyloid formation. Biochimica et Biophysica Acta (BBA) - Proteins and Proteomics, 1867(3),
344-349.
Duan, W., Richardson, S.J., Köhrle, J., Chang, L., Southwell, B.R., Harms, P.J., Brack, C.M., Pettersson, T.M., &
Schreiber. G. (1995). Binding of thyroxine to pig transthyretin, its cDNA structure, and other properties.
European Journal of Biochemistry, 230(3), 977-986.
Ferreira, N., Saraiva, M.J., & Almeida, M.R. (2019). Uncovering the neuroprotective mechanisms of curcumin on
transthyretin amyloidosis. International Journal of Molecular Sciences, 20(6), 1287.
Garcia-Alloza, M., Borrelli L.A., Rozkalne, A., Hyman B.T., & Bacskai, B.J. (2007). Curcumin labels amyloid
pathology in vivo, disrupts existing plaques, and partially restores distorted neurites in an Alzheimer
mouse model. Journal of Neurochemistry, 102(4), 1095-1104.
Holmgren, G., Ericzon, B.G., Groth, C.G., Steen, L., Suhr, O., Andersen, O., Wallin, B.G., Seymour, A., Richardson,
S., Hawkins, P.N., & Pepys, M.B. (1993). Clinical improvement and amyloid regression after liver
transplantation in hereditary transthyretin amyloidosis. The Lancet, 341(8853), 1113-1116.
Hou, X., Richardson, S.J., Aguilar, M-I., & Small, D.H. (2005). Binding of amyloidogenic transthyretin to the plasma
membrane alters membrane fluidity and induces neurotoxicity. Biochemistry, 44(34), 11618-11627.
Hund, E., Linke, R.P., Willig, F. & Gra, A. (2001). Transthyretin-associated neuropathic amyloidosis. Pathogenesis
and treatment. Neurology, 56(4), 431-435.
Jacobson, D.R., & Buxaum, J.N. (1991). Genetic aspects of amyloidosis. Advances in Human Genetics, 20,
69-123.
Kaewmeechai, S., Poodproh, R., & Prapunpoj P. (2019). Transthyretin variant in Thai people is likely to associate
with pathogenesis. Rajamangala University of Technology Srivijaya Research Journal, 11(3), 387-401.
Kawas, C.H. (2006). Medications and diet protective factors for AD?. Alzheimer Disease & Associated Disorders,
20(3 suppl 2), S89–S96.
Lashuel, H.A., Lai, Z., & Kelly, J.W. (1998). Characterization of the transthyretin acid denaturation pathways by
analytical ultracentrifugation: implications for wild-type, V30M, and L55P amyloid fibril formation.
Biochemistry Journal, 37(51), 17851-17864.
Park, G.Y., Jamerlan, A., Shim, K.H., & An, S.S.A. (2019). Diagnostic and treatment approaches involving
Transthyretin in amyloidogenic diseases. International Journal of Molecular Sciences, 20(12), 2982-2998.
Peterson, S.A., Klabunde, T., Lashuel, H.A., Purkey, H., Sacchettini, J.C., & Kelly, J.W. (1998). Inhibiting
transthyretin conformational changes that lead to amyloid fibril formation. Proceedings of the National
Academy of Sciences of the United States of America, 95(22), 12956-12960.
Prapunpoj, P., Richardson, S.J., & Schreiber, G. (2002). Crocodile transthyretin: structure, function, and evolution.
American Journal of Physiology-Regulatory, Integrative and Comparative Physiology, 283(4),
R885–R896.
Prapunpoj, P., Yamauchi, K., Nishiyama, N., Richardson, S.J., & Schreiber, G. (2000). Evolution of structure,
ontogeny of gene expression, and function of Xenopus laevis transthyretin. American Journal of
Physiology-Regulatory, Integrative and Comparative Physiology, 279, R2026-R204.
Reixach, N., Deechongkit, S., Jiang, X., Kelly, J.W., & Buxbaum, J.N. (2004). Tissue damage in the amyloidoses:
Transthyretin monomers and nonnative oligomers are the major cytotoxic species in tissue culture.
Proceedings of the National Academy of Sciences of the United States of America, 101(9), 2817-2822.
Richardson, S.J. (2007). Cell and molecular biology of transthyretin and thyroid hormones. International Review of
Cytology, 258, 137-193.
Ruberg, F.L. & Berk, J.L. (2012). Transthyretin (TTR) cardiac amyloidosis. Circulation, 126(10), 1286-1300.
Saelices, L., Johnson, L.M., Liang, W.Y., Sawaya, M.R., Cascio, Duilio, Ruchala, P., Whitelegge, J., Jiang, L., Riek,
R., & Eisenberg, D.S. (2015). Uncovering the mechanism of aggregation of human transthyretin. Journal
of Biological Chemistry, 290(48), 28932-28943.
Sorgjerd, K., Klingstedt, T., Lindgren, M., Kagedal, K., & Hammarstrom, P. (2008). Prefibrillar transthyretin
oligomers and cold stored native tetrameric transthyretin are cytotoxic in cell culture. Biochemical and
Biophysical Research Communications, 377(4), 1072-1078.
Sousa, M.M., & Saraiva, M. (2003). Neurodegeneration in familial amyoloid polyneuropathy: from pathology to
molecular signaling. Progress in Neurobiology, 71(5), 385-400.
Tan, S.Y., Pepys, M.B., & Hawkins, P.N. (1995). Treatment of amyloidosis. American Journal of Kidney Diseases,
26(2), 267-285.
Tola, A.J, Leelawatwattana, L., & Prapunpoj, P. (2019). The catalytic kinetics of chicken transthyretin towards
human A1-42. Comparative Biochemistry and Physiology Part C: Toxicology & Pharmacology, 226,
doi: 10.1016/j.cbpc.2019.108610.
Vogl, T., & Glieder, A. (2013). Regulation of Pichia pastoris promoters and its consequences for protein
production. New Biotechnology, 30(4), 385-404.
Wang, X., Cattaneo, F., Ryno, L., Hulleman, J., Reixach, N., & Buxbaum, J. (2014). The systemic amyloid precursor
transthyretin (TTR) behaves as a neuronal stress protein regulated by HSF1 in SH-SY5Y human
neuroblastoma cells and APP23 Alzheimer’s disease model mice. The Journal of Neuroscience, 34(21),
7253-7265.
Xue, Q., Zheng, Q.C., Zhang, J.L., Cui, Y.L. Chu, W.T., & Zhang, H.X. (2014). Mutation and low pH effect on the
stability as well as unfolding kinetics of transthyretin dimer. Biophysical Chemistry, 189, 8-15.
Yang, M., Lei, M., & Huo, S. (2003). Why is Leu55→Pro55 transthyretin variant the most amyloidgenic; Insights
from molecular dynamics simulations of transthyretin monomers. Protein Science, 12(6), 1222-1231.
Yiannopoulou, K.G., & Papageorgiou, S.G. (2013). Current and future treatments for Alzheimer’s disease.
Therapeutic Advances in Neurological Disorders, 6(1), 19-33.
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2021-02-15
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