Abstract
Protein aggregation and inclusion body formation have been a key causal phenomenon behind a majority of neurodegenerative disorders. Various approaches aimed at preventing the formation/elimination of protein aggregates are being developed to control these diseases. Molecular chaperones are a class of protein that not only direct the functionally relevant fold of the protein but also perform quality control against stress, misfolding/aggregation. Genes that encode molecular chaperones are induced and expressed in response to extreme stress conditions to “salvage” the cell by the “unfolded protein response” (UPR) signaling pathway. Here we describe in detail the various in vitro and in vivo assays involved in identifying the chaperone activity of proteins using human calnuc as a model protein. Calnuc is a Golgi resident, calcium-binding protein, identified as chaperone protein and is reported to protect the cells against the cytotoxicity caused by amyloidosis and ER stress. Calnuc is also reported to regulate Gαi activity and inflammation apart from the role of chaperoning against amyloid proteins.
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References
Reiss AB, Arain HA, Stecker MM, Siegart NM, Kasselman LJ (2018) Amyloid toxicity in Alzheimer's disease. Rev Neurosci 29(6):613–627. https://doi.org/10.1515/revneuro-2017-0063
Eisele YS, Monteiro C, Fearns C, Encalada SE, Wiseman RL, Powers ET, Kelly JW (2015) Targeting protein aggregation for the treatment of degenerative diseases. Nat Rev Drug Discov 14:759–780. https://doi.org/10.1038/nrd4593
Ross CA, Poirier MA (2004) Protein aggregation and neurodegenerative disease. Nat Med 10(Suppl):S10–S17. https://doi.org/10.1038/nm1066
Liberek K, Lewandowska A, Zietkiewicz S (2008) Chaperones in control of protein disaggregation. EMBO J 27:328–335. https://doi.org/10.1038/sj.emboj.7601970
Stull F, Koldewey P, Humes JR, Radford SE, Bardwell JCA (2016) Substrate protein folds while it is bound to the ATP-independent chaperone Spy. Nat Struct Mol Biol 23:53–58. https://doi.org/10.1038/nsmb.3133
Beissinger M, Buchner J (1998) How chaperones fold proteins. Biol Chem 379:245
Schroder M, Kaufman RJ (2005) ER stress and the unfolded protein response. Mutat Res 569:29–63. https://doi.org/10.1016/j.mrfmmm.2004.06.056
Liu CY, Kaufman RJ (2003) The unfolded protein response. J Cell Sci 116:1861–1862. https://doi.org/10.1242/jcs.00408
Haslbeck M, Buchner J (2015) Assays to characterize molecular chaperone function in vitro. Methods Mol Biol 1292:39–51. https://doi.org/10.1007/978-1-4939-2522-3_3
Glover JR, Lindquist S (1998) Hsp104, Hsp70, and Hsp40: a novel chaperone system that rescues previously aggregated proteins. Cell 94:73–82
Nollen EA, Brunsting JF, Roelofsen H, Weber LA, Kampinga HH (1999) In vivo chaperone activity of heat shock protein 70 and thermotolerance. Mol Cell Biol 19:2069–2079
Mymrikov EV, Daake M, Richter B, Haslbeck M, Buchner J (2017) The chaperone activity and substrate spectrum of human small heat shock proteins. J Biol Chem 292:672–684. https://doi.org/10.1074/jbc.M116.760413
Dehle FC, Ecroyd H, IF M, Carver JA (2010) alphaB-Crystallin inhibits the cell toxicity associated with amyloid fibril formation by kappa-casein and the amyloid-beta peptide. Cell Stress Chaperones 15:1013–1026. https://doi.org/10.1007/s12192-010-0212-z
Kanuru M, Raman R, Aradhyam GK (2013) Serine protease activity of calnuc: regulation by Zn2+ and G proteins. J Biol Chem 288:1762–1773. https://doi.org/10.1074/jbc.M112.382846
Long F, Cho W, Ishii Y (2011) Expression and purification of 15N- and 13C-isotope labeled 40-residue human Alzheimer's beta-amyloid peptide for NMR-based structural analysis. Protein Expr Purif 79:16–24. https://doi.org/10.1016/j.pep.2011.05.012
Bergmeyer HU (1974) Methods of enzymatic analysis. Verlag Chemie; Academic Press, Weinheim
Lindberg DJ, Wenger A, Sundin E, Wesen E, Westerlund F, Esbjorner EK (2017) Binding of Thioflavin-T to Amyloid Fibrils Leads to Fluorescence Self-Quenching and Fibril Compaction. Biochemistry 56:2170–2174. https://doi.org/10.1021/acs.biochem.7b00035
Gras SL, Waddington LJ, Goldie KN (2011) Transmission electron microscopy of amyloid fibrils. Methods Mol Biol 752:197–214. https://doi.org/10.1007/978-1-60327-223-0_13
El-Agnaf OM, Jakes R, Curran MD, Middleton D, Ingenito R, Bianchi E, Pessi A, Neill D, Wallace A (1998) Aggregates from mutant and wild-type alpha-synuclein proteins and NAC peptide induce apoptotic cell death in human neuroblastoma cells by formation of beta-sheet and amyloid-like filaments. FEBS Lett 440:71–75
Kanuru M, Aradhyam GK (2017) Chaperone-like Activity of Calnuc Prevents Amyloid Aggregation. Biochemistry 56:149–159. https://doi.org/10.1021/acs.biochem.6b00660
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Vignesh, R., Aradhyam, G.K. (2019). Chaperoning Against Amyloid Aggregation: Monitoring In Vitro and In Vivo. In: Heizmann, C. (eds) Calcium-Binding Proteins of the EF-Hand Superfamily. Methods in Molecular Biology, vol 1929. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-9030-6_10
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DOI: https://doi.org/10.1007/978-1-4939-9030-6_10
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