An optimized Western blot assay provides a comprehensive assessment of the physiological endoproteolytic processing of the prion protein
2022; Elsevier BV; Volume: 299; Issue: 2 Linguagem: Inglês
10.1016/j.jbc.2022.102823
ISSN1083-351X
AutoresIlaria Vanni, Floriana Iacobone, Claudia D’Agostino, Matteo Giovannelli, Laura Pirisinu, Hermann C. Altmeppen, Joaquı́n Castilla, Juan María Torres, Umberto Agrimi, Romolo Nonno,
Tópico(s)Trace Elements in Health
ResumoThe prion protein (PrPC) is subjected to several conserved endoproteolytic events producing bioactive fragments that are of increasing interest for their physiological functions and their implication in the pathogenesis of prion diseases and other neurodegenerative diseases. However, systematic and comprehensive investigations on the full spectrum of PrPC proteoforms have been hampered by the lack of methods able to identify all PrPC-derived proteoforms. Building on previous knowledge of PrPC endoproteolytic processing, we thus developed an optimized Western blot assay able to obtain the maximum information about PrPC constitutive processing and the relative abundance of PrPC proteoforms in a complex biological sample. This approach led to the concurrent identification of the whole spectrum of known endoproteolytic-derived PrPC proteoforms in brain homogenates, including C-terminal, N-terminal and, most importantly, shed PrPC-derived fragments. Endoproteolytic processing of PrPC was remarkably similar in the brain of widely used wild type and transgenic rodent models, with α-cleavage-derived C1 representing the most abundant proteoform and ADAM10-mediated shedding being an unexpectedly prominent proteolytic event. Interestingly, the relative amount of shed PrPC was higher in WT mice than in most other models. Our results indicate that constitutive endoproteolytic processing of PrPC is not affected by PrPC overexpression or host factors other than PrPC but can be impacted by PrPC primary structure. Finally, this method represents a crucial step in gaining insight into pathophysiological roles, biomarker suitability, and therapeutic potential of shed PrPC and for a comprehensive appraisal of PrPC proteoforms in therapies, drug screening, or in the progression of neurodegenerative diseases. The prion protein (PrPC) is subjected to several conserved endoproteolytic events producing bioactive fragments that are of increasing interest for their physiological functions and their implication in the pathogenesis of prion diseases and other neurodegenerative diseases. However, systematic and comprehensive investigations on the full spectrum of PrPC proteoforms have been hampered by the lack of methods able to identify all PrPC-derived proteoforms. Building on previous knowledge of PrPC endoproteolytic processing, we thus developed an optimized Western blot assay able to obtain the maximum information about PrPC constitutive processing and the relative abundance of PrPC proteoforms in a complex biological sample. This approach led to the concurrent identification of the whole spectrum of known endoproteolytic-derived PrPC proteoforms in brain homogenates, including C-terminal, N-terminal and, most importantly, shed PrPC-derived fragments. Endoproteolytic processing of PrPC was remarkably similar in the brain of widely used wild type and transgenic rodent models, with α-cleavage-derived C1 representing the most abundant proteoform and ADAM10-mediated shedding being an unexpectedly prominent proteolytic event. Interestingly, the relative amount of shed PrPC was higher in WT mice than in most other models. Our results indicate that constitutive endoproteolytic processing of PrPC is not affected by PrPC overexpression or host factors other than PrPC but can be impacted by PrPC primary structure. Finally, this method represents a crucial step in gaining insight into pathophysiological roles, biomarker suitability, and therapeutic potential of shed PrPC and for a comprehensive appraisal of PrPC proteoforms in therapies, drug screening, or in the progression of neurodegenerative diseases. The cellular prion protein (PrPC) is mainly known for its pivotal role in transmissible spongiform encephalopathies (TSEs), or prion diseases, which are fatal neurodegenerative diseases affecting humans and animals. These unusual diseases are associated with the central nervous system accumulation of autocatalytically self-replicating aggregates of PrPSc, a misfolded pathological isoform of the host-encoded PrPC (1Prusiner S.B. Novel proteinaceous infectious particles cause scrapie.Science. 1982; 216: 136-144Crossref PubMed Scopus (4248) Google Scholar). PrPC is a cell surface glycoprotein of 209 amino acids, encoded by the Prnp gene. Despite the translation of a single polypeptide, the protein undergoes several post-translational modifications, that is, the addition of N-linked sugars at residues 181 and 197, a glycophosphatidylinositol (GPI) anchor for membrane attachment and a disulphide bond (2Stahl N. Borchelt D.R. Hsiao K. Prusiner S.B. 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C-terminal peptides modelling constitutive PrPC processing demonstrate ameliorated toxicity predisposition consequent to alpha-cleavage.Biochem. J. 2014; 459: 103-115Crossref PubMed Scopus (11) Google Scholar). A C2-like fragment has been reported to be the main cleavage product of PrPSc in the brain of Creutzfeldt–Jakob disease patients (10Chen S.G. Teplow D.B. Parchi P. Teller J.K. Gambetti P. Autilio-Gambetti L. Truncated forms of the human prion protein in normal brain and in prion diseases.J. Biol. Chem. 1995; 270: 19173-19180Abstract Full Text Full Text PDF PubMed Scopus (458) Google Scholar, 25Jimenez-Huete A. Lievens P.M. Vidal R. Piccardo P. Ghetti B. Tagliavini F. et al.Endogenous proteolytic cleavage of normal and disease-associated isoforms of the human prion protein in neural and non-neural tissues.Am. J. Pathol. 1998; 153: 1561-1572Abstract Full Text Full Text PDF PubMed Scopus (156) Google Scholar) and in prion-infected neuroblastoma cells (28Caughey B. Raymond G.J. 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In physiological conditions, this extreme C-terminal cleavage is implicated in PrPC homeostasis, but it has also been suggested that shed PrPC holds some own independent functions as a soluble trophic factor acting on close or distant cells as well as a neuroprotective fragment in the extracellular space (19Altmeppen H.C. Puig B. Dohler F. Thurm D.K. Falker C. Krasemann S. et al.Proteolytic processing of the prion protein in health and disease.Am. J. Neurodegener. Dis. 2012; 1: 15-31PubMed Google Scholar, 36Altmeppen H.C. Prox J. Puig B. Kluth M.A. Bernreuther C. Thurm D. et al.Lack of a-disintegrin-and-metalloproteinase ADAM10 leads to intracellular accumulation and loss of shedding of the cellular prion protein in vivo.Mol. Neurodegener. 2011; 6: 36Crossref PubMed Scopus (89) Google Scholar). A conceivable dual role has been postulated for PrP shedding during prion diseases. 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