फोटो प्रेरित पार से जोड़ने असंशोधित प्रोटीन (पीआईसीयूपी) Amyloidogenic पेप्टाइड्स लागू करने के लिए

Summary

फोटो प्रेरित पार से जोड़ने असंशोधित प्रोटीन (पीआईसीयूपी) के metastable प्रोटीन मिश्रण में oligomer आकार के वितरण के लक्षण वर्णन की अनुमति देता है. हम तीन प्रतिनिधि amyloidogenic 40 पेप्टाइड्स पीआईसीयूपी के आवेदन का प्रदर्शन – और 42-β प्रोटीन amyloid और कैल्सीटोनिन के अवशेषों रूपों, और एक नियंत्रण पेप्टाइड वृद्धि हार्मोन जारी कारक है.

Abstract

The assembly of amyloidogenic proteins into toxic oligomers is a seminal event in the pathogenesis of protein misfolding diseases, including Alzheimer’s, Parkinson’s, and Huntington’s diseases, hereditary amyotrophic lateral sclerosis, and type 2 diabetes. Owing to the metastable nature of these protein assemblies, it is difficult to assess their oligomer size distribution quantitatively using classical methods, such as electrophoresis, chromatography, fluorescence, or dynamic light scattering. Oligomers of amyloidogenic proteins exist as metastable mixtures, in which the oligomers dissociate into monomers and associate into larger assemblies simultaneously. PICUP stabilizes oligomer populations by covalent cross-linking and when combined with fractionation methods, such as sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) or size-exclusion chromatography (SEC), PICUP provides snapshots of the oligomer size distributions that existed before cross-linking. Hence, PICUP enables visualization and quantitative analysis of metastable protein populations and can be used to monitor assembly and decipher relationships between sequence modifications and oligomerization¹. Mechanistically, PICUP involves photo-oxidation of Ru²⁺ in a tris(bipyridyl)Ru(II) complex (RuBpy) to Ru³⁺ by irradiation with visible light in the presence of an electron acceptor. Ru³⁺ is a strong one-electron oxidizer capable of abstracting an electron from a neighboring protein molecule, generating a protein radical^1,2. Radicals are unstable, highly-reactive species and therefore disappear rapidly through a variety of intra- and intermolecular reactions. A radical may utilize the high energy of an unpaired electron to react with another protein monomer forming a dimeric radical, which subsequently loses a hydrogen atom and forms a stable, covalently-linked dimer. The dimer may then react further through a similar mechanism with monomers or other dimers to form higher-order oligomers. Advantages of PICUP relative to other photo- or chemical cross-linking methods^3,4 include short (≤1 s) exposure to non-destructive visible light, no need for pre facto modification of the native sequence, and zero-length covalent cross-linking. In addition, PICUP enables cross-linking of proteins within wide pH and temperature ranges, including physiologic parameters. Here, we demonstrate application of PICUP to cross-linking of three amyloidogenic proteins the 40- and 42-residue amyloid β-protein variants (Aβ40 and Aβ42), and calcitonin, and a control protein, growth-hormone releasing factor (GRF).

Protocol

1. पेप्टाइड तैयारी ~ Lyophilized पेप्टाइड के 100-200 μg और लेबल, सिलिकॉन लेपित, कम adsorbent microfuge ट्यूबों में Microbalance हस्तांतरण का उपयोग कर वजन. यहाँ, हम Aβ40 के मानव दृश्यों, Aβ42, कैल्सीटोनिन, और GRF का उपयोग करें. यहाँ, हम पूर्व 1…

Discussion

पीआईसीयूपी मूल स्थिर प्रोटीन परिसरों ² अध्ययन के लिए विकसित किया गया था. विधि metastable amyloid प्रोटीन सहित ¹⁰ Aβ, और रोग संबद्ध prion PRP ^{11 एससी,} और α-synuclein ¹² विधानसभाओं, मात्रात्मक अध्ययन के बाद लागू किया गया था. सबस…

Materials

Material Name	Tipo	Company	Catalogue Number	Comment
Dolan-Jenner 200-W incandescent lamp	Altro	Dolan-Jenner Industries	Model 170-D	The heat generated by the lamp does not affect samples for short incubation periods.
35-mm SLR camera body	Tool	Pentax	SP500 model	In our settings, a bellows is attached to the body of the camera to provide a convenient chamber for irradiation of the sample placed 10 cm away from the light source.
Clear, thin walled PCR tubes	Altro	Eppendorf	951010006 supplied by Fisher L22-003-24
Glass vials (1.8 mL)	Altro	Kimble Chromatography	60940A 2, supplied by Fisher 03-340-60
GRF	Reagent	Bachem	H-3695
HFIP	Reagent	TCI America	H0424	Use in a fume hood.
Aβ40 and Aβ42	Reagent	UCLA Biopolymers Laboratory
Calcitonin	Reagent	American Peptide	22-1-10
Tris(2,2-bipridyl)dichlororuthenium(II) hexahydrate	Reagent	Sigma	224758-1G	Vortex until the solution is clear. Cover the RuBpy tube with foil to protect the reagent from ambient light. RuBpy is prepared freshly each time and should be used within 48 h.
Ammonium persulfate	Reagent	Sigma	A-7460	Vortex until the solution is clear. APS is prepared freshly each time and should be used within 48 h.
β-mercaptoethanol	Reagent	Sigma	M7154-25 ML	Can be used when SDS-PAGE analysis is performed.
Novex Tricine SDS Sample Buffer (2x)	Reagent	Invitrogen	LC1676
XCell SureLock Mini-Cell	Tool	Invitrogen	EI0001
Novex Tricine Gels (10-20%)	Altro	Invitrogen	EC6625B0X
Novex Tricine SDS Running Buffer (10x )		Invitrogen	LC1675
Silver Express Staining Kit		Invitrogen	LC6100