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1.
Protein Sci ; 28(5): 889-899, 2019 05.
Article in English | MEDLINE | ID: mdl-30825227

ABSTRACT

Amyloid-ß peptides (Aßs) are generated in a membrane-embedded state by sequential processing of amyloid precursor protein (APP). Although shedding of membrane-embedded Aß is essential for its secretion and neurotoxicity, the mechanism behind shedding regulation is not fully elucidated. Thus, we devised a Langmuir film balance-based assay to uncover this mechanism. We found that Aß shedding was enhanced under acidic pH conditions and in lipid compositions resembling raft microdomains, which are directly related to the microenvironment of Aß generation. Furthermore, Aß shedding efficiency was determined by the length of the C-terminal membrane-spanning region, whereas pH responsiveness appears to depend on the N-terminal ectodomain. These findings indicate that Aß shedding may be directly coupled to its generation and represents an unrecognized control mechanism regulating the fate of membrane-embedded products of APP processing.


Subject(s)
Amyloid beta-Peptides/chemistry , Cell-Derived Microparticles/metabolism , Endosomes/chemistry , Amyloid beta-Peptides/metabolism , Animals , Cell-Derived Microparticles/chemistry , Circular Dichroism , Endosomes/metabolism , Humans , Hydrogen-Ion Concentration , Membrane Microdomains/metabolism , Protein Domains
2.
Sci Rep ; 8(1): 1494, 2018 01 24.
Article in English | MEDLINE | ID: mdl-29367639

ABSTRACT

The causal relationship between conformational folding and disulfide bonding in protein oxidative folding remains incompletely defined. Here we show a stage-dependent interplay between the two events in oxidative folding of C-reactive protein (CRP) in live cells. CRP is composed of five identical subunits, which first fold spontaneously to a near-native core with a correctly positioned C-terminal helix. This process drives the formation of the intra-subunit disulfide bond between Cys36 and Cys97. The second stage of subunit folding, however, is a non-spontaneous process with extensive restructuring driven instead by the intra-subunit disulfide bond and guided by calcium binding-mediated anchoring. With the folded subunits, pentamer assembly ensues. Our results argue that folding spontaneity is the major determinant that dictates which event acts as the driver. The stepwise folding pathway of CRP further suggests that one major route might be selected out of the many in theory for efficient folding in the cellular environment.


Subject(s)
C-Reactive Protein/chemistry , Disulfides/chemistry , Protein Conformation , Protein Folding , Humans , Models, Molecular , Oxidation-Reduction
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