घुलनशील और अघुलनशील पीआरपी oligomers के सामान्य मानव मस्तिष्क में अलगाव

Summary

सेलुलर prion प्रोटीन की एक नई प्रजाति (पीआरपी^सी) हाल ही में यहाँ वर्णित तरीकों का उपयोग कर असंक्रमित मानव दिमाग में पहचान की गई है. इन तरीकों को विभिन्न पीआरपी प्रजातियों को अलग करने के लिए इस्तेमाल किया जा सकता है, जबकि उनमें से कुछ भी हैं मानव दिमाग से अलग अन्य misfolded प्रोटीन समुच्चय में उपयोगी हैं.

Abstract

The central event in the pathogenesis of prion diseases involves a conversion of the host-encoded cellular prion protein PrP^C into its pathogenic isoform PrP^{Sc 1}. PrP^C is detergent-soluble and sensitive to proteinase K (PK)-digestion, whereas PrP^Sc forms detergent-insoluble aggregates and is partially resistant to PK^2-6. The conversion of PrP^C to PrP^Sc is known to involve a conformational transition of α-helical to β-sheet structures of the protein. However, the in vivo pathway is still poorly understood. A tentative endogenous PrP^Sc, intermediate PrP* or “silent prion”, has yet to be identified in the uninfected brain⁷.

Using a combination of biophysical and biochemical approaches, we identified insoluble PrP^C aggregates (designated iPrP^C) from uninfected mammalian brains and cultured neuronal cells^{8, 9}. Here, we describe detailed procedures of these methods, including ultracentrifugation in detergent buffer, sucrose step gradient sedimentation, size exclusion chromatography, iPrP enrichment by gene 5 protein (g5p) that specifically bind to structurally altered PrP forms¹⁰, and PK-treatment. The combination of these approaches isolates not only insoluble PrP^Sc and PrP^C aggregates but also soluble PrP^C oligomers from the normal human brain. Since the protocols described here have been used to isolate both PrP^Sc from infected brains and iPrP^C from uninfected brains, they provide us with an opportunity to compare differences in physicochemical features, neurotoxicity, and infectivity between the two isoforms. Such a study will greatly improve our understanding of the infectious proteinaceous pathogens. The physiology and pathophysiology of iPrP^C are unclear at present. Notably, in a newly-identified human prion disease termed variably protease-sensitive prionopathy, we found a new PrP^Sc that shares the immunoreactive behavior and fragmentation with iPrP^{C 11, 12}. Moreover, we recently demonstrated that iPrP^C is the main species that interacts with amyloid-β protein in Alzheimer disease¹³. In the same study, these methods were used to isolate Abeta aggregates and oligomers in Alzheimer’s disease¹³, suggesting their application to non-prion protein aggregates involved in other neurodegenerative disorders.

Protocol

1. मस्तिष्क Homogenate और डिटर्जेंट (S2) घुलनशील और अघुलनशील Fractions (P2) की तैयारी 100 मिलीग्राम जमे हुए मानव मस्तिष्क के ऊतकों लो और 100 μl lysis बफर (10 मिमी Tris, 150 मिमी NaCl, 0.5% P-40 Nonidet (एनपी 40), 0.5% EDTA, 7.4 पीएच). यह एक ताररहित मोटर द्वार?…

Discussion

दृष्टिकोण के संयोजन यहाँ की सूचना लगातार अघुलनशील पीआरपी ^सी और सामान्य मानव मस्तिष्क से समुच्चय घुलनशील पीआरपी ^सी oligomers आइसोलेट्स. एक घंटे के लिए 100.000 XG ultracentrifugation एक क्लासिक तरीका है कि व्यापक रूप स?…

Materials

Name of the reagent	Company	Catalogue number	Comments (optional)
Phenylmethylsulfonyl fluoride	Sigma-Aldrich	P7626	72 mM in 2-propanol
Proteinase K	Sigma-Aldrich	P2308	2 mg/ml in H2O
PNGase F	New England Biolabs	MWGF200
Ultracentrifuge LE-80K, SW55 rotor	Beckman Coulter	Part No. 365668, 356860
Superdex 200 HR beads	GE Healthcare	17-1088-01
AKTA FPLC system	GE Healthcare	18-1900-26
Molecular weight markers	Sigma-Aldrich	MWGF200	Varied
Dynabeads M-280 magnetic beads	Invitrogen	143-01
3F4 antibody	Covance	SIG-39600
1E4 antibody	Cell Sciences	M1840
Ready gel 15% Tris-HCl pre-cast gels	Bio-Rad	345-0020