Header

UZH-Logo

Maintenance Infos

Modifiers of prion protein biogenesis and recycling identified by a highly-parallel endocytosis kinetics assay


Ballmer, Boris A; Moos, Rita; Liberali, Prisca; Pelkmans, Lucas; Hornemann, Simone; Aguzzi, Adriano (2017). Modifiers of prion protein biogenesis and recycling identified by a highly-parallel endocytosis kinetics assay. Journal of Biological Chemistry, 292(20):8356-8368.

Abstract

The cellular prion protein, PrPC, is attached by a glycosylphosphatidylinositol anchor to the outer leaflet of the plasma membrane. Its misfolded isoform PrPSc is the causative agent of prion diseases. Conversion of PrPC into PrPSc is thought to take place at the cell surface or in endo-lysosomal organelles. Understanding the intracellular trafficking of PrPC may therefore help elucidating the conversion process. Here we describe a time-resolved fluorescence resonance energy transfer (FRET) assay reporting membrane expression and real-time internalization rates of PrPC The assay is suitable for high-throughput genetic and pharmaceutical screens for modulators of PrPC trafficking. Simultaneous administration of FRET donor and acceptor anti-PrPC antibodies to living cells yielded a measure of PrPC surface density, whereas sequential addition of each antibody visualized the internalization rate of PrPC (Z'-factor > 0.5). RNA interference assays showed that suppression of AP2M1, RAB5A, VPS35 and M6PR blocked PrPC internalization, whereas downregulation of GIT2 and VPS28 increased it. PrPC cell surface expression was reduced by downregulation of RAB5A, VPS28 and VPS35, and enhanced by silencing EHD1. These data identify a network of proteins implicated in PrPC trafficking, and demonstrates the power of this assay for identifying modulators of PrPC trafficking.

Abstract

The cellular prion protein, PrPC, is attached by a glycosylphosphatidylinositol anchor to the outer leaflet of the plasma membrane. Its misfolded isoform PrPSc is the causative agent of prion diseases. Conversion of PrPC into PrPSc is thought to take place at the cell surface or in endo-lysosomal organelles. Understanding the intracellular trafficking of PrPC may therefore help elucidating the conversion process. Here we describe a time-resolved fluorescence resonance energy transfer (FRET) assay reporting membrane expression and real-time internalization rates of PrPC The assay is suitable for high-throughput genetic and pharmaceutical screens for modulators of PrPC trafficking. Simultaneous administration of FRET donor and acceptor anti-PrPC antibodies to living cells yielded a measure of PrPC surface density, whereas sequential addition of each antibody visualized the internalization rate of PrPC (Z'-factor > 0.5). RNA interference assays showed that suppression of AP2M1, RAB5A, VPS35 and M6PR blocked PrPC internalization, whereas downregulation of GIT2 and VPS28 increased it. PrPC cell surface expression was reduced by downregulation of RAB5A, VPS28 and VPS35, and enhanced by silencing EHD1. These data identify a network of proteins implicated in PrPC trafficking, and demonstrates the power of this assay for identifying modulators of PrPC trafficking.

Statistics

Altmetrics

Downloads

7 downloads since deposited on 05 Apr 2017
7 downloads since 12 months
Detailed statistics

Additional indexing

Item Type:Journal Article, refereed, original work
Communities & Collections:04 Faculty of Medicine > University Hospital Zurich > Institute of Neuropathology
Dewey Decimal Classification:570 Life sciences; biology
610 Medicine & health
Language:English
Date:24 March 2017
Deposited On:05 Apr 2017 10:07
Last Modified:06 Aug 2017 04:20
Publisher:American Society for Biochemistry and Molecular Biology
ISSN:0021-9258
Free access at:PubMed ID. An embargo period may apply.
Publisher DOI:https://doi.org/10.1074/jbc.M116.773283
PubMed ID:28341739

Download

Preview Icon on Download
Preview
Content: Accepted Version
Filetype: PDF
Size: 3MB
View at publisher

TrendTerms

TrendTerms displays relevant terms of the abstract of this publication and related documents on a map. The terms and their relations were extracted from ZORA using word statistics. Their timelines are taken from ZORA as well. The bubble size of a term is proportional to the number of documents where the term occurs. Red, orange, yellow and green colors are used for terms that occur in the current document; red indicates high interlinkedness of a term with other terms, orange, yellow and green decreasing interlinkedness. Blue is used for terms that have a relation with the terms in this document, but occur in other documents.
You can navigate and zoom the map. Mouse-hovering a term displays its timeline, clicking it yields the associated documents.

Author Collaborations