This volume is divided in six section covering the most experimental approaches involved in the study of the unfolded protein response (UPR) pathway. Chapters detail determination of unfolded protein levels, methods to study UPR signal transmission, analysing the outcomes of the UPR pathway activation, UPR studies in mammalian models, UPR in alternative models, and UPR and disease. Written in the format of the highly successful Methods in Molecular Biology series, each chapter includes an introduction to the topic, lists necessary materials and reagents, includes tips on troubleshooting and known pitfalls, and step-by-step, readily reproducible protocols.
Authoritative and cutting-edge, The Unfolded Protein Response: Methods and Protocols aims to describe key methods and approaches used in the study of the UPR pathway and its complex cellular implications.
Chapter 6 is available open access under a Creative Commons Attribution 4.0 International License via link.springer.com.
Measuring cysteine exposure in unfolded proteins with tetraphenylethene maleimide and its analogues.- Fluorescence-based biosensors for the detection of the unfolded protein response.- Indirect methods to measure unfolded proteins in living cells using fluorescent proteins.- A platform technology for monitoring the Unfolded Protein Response.- The use of fluorescent protein fusions to monitor the unfolded protein response and protein foldase-substrate interactions in plant protoplasts.- High-throughput analysis of protein turnover with tandem fluorescent protein timers.- Detection of HAC1 mRNA Splicing by RT-PCR in
Saccharomyces cerevisiae.- Determination of protein translation during the activation of the UPR.- Determination of the stability and intracellular (intra-nuclear) targeting and recruitment of pre-HAC1 mRNA in the Saccharomyces cerevisiae during the activation of UPR.- Assays to study IRE1 activation and signaling.- Analysis of XBP1 contribution to hyperosmolarity-induced lipid synthesis.- Integrating an ER stress reporter for monitoring genome wide UPR-ER in budding yeast.- Analyze mouse knockout models of UPR pathway elements.-
In vitro stimulation of IRE1a/XBP1-deficient B cells with LPS.- Detection of PERK signaling in the central nervous system.- Methods to study the mitochondrial unfolded protein response (UPRmt)
in Caenorhabditis elegans.- Drosophila Unfolded Protein Response (UPR) assays
in vitro and
in vivo.- Protein preparation for proteomic analysis of the unfolded protein response in
Arabidopsis thaliana.- Structure-based drug discovery of IRE1 modulators.- Chemical screening to detect ER stress regulators.
Inhaltsverzeichnis
Part I Determination of unfolded protein levels
1 Measuring cysteine exposure in unfolded proteins with tetraphenylethene maleimide and its analogues
Shouxiang Zhang and Yuning Hong
2 Fluorescence-based biosensors for the detection of the unfolded protein response
Heinrich Kroukamp, Kai Peng, Ian T. Paulsen, and Riaan den Haan
3 Indirect methods to measure unfolded proteins in living cells using fluorescent proteins
Elisabet Navarro-Tapia, and Roberto Pérez-Torrado
4 A platform technology for monitoring the Unfolded Protein Response
Bhagyashree Bachhav, Carlos A. Origel Marmolejo, Yafet Arefeayne, and Laura Segatori
5 The use of fluorescent protein fusions to monitor the unfolded protein response and protein foldase-substrate interactions in plant protoplasts
Rina Carrillo, Elizabeth Feldeverd, and David A. Christopher
Part II Methods to study UPR signal transmission
6 High-throughput analysis of protein turnover with tandem fluorescent protein timers
Jia Jun Fung, Karla Blöcher-Juárez, and Anton Khmelinskii
7 Detection of HAC1 mRNA Splicing by RT-PCR in Saccharomyces cerevisiae
Jagadeesh Kumar Uppala and Madhusudan Dey
Part III Analysing the outcomes of the UPR pathway activation
8 Determination of protein translation during the activation of the UPR
Weihan Li and Robert H. Singer
9 Determination of the stability and intracellular (intra-nuclear) targeting and recruitment of pre-HAC1 mRNA in the Saccharomyces cerevisiae during the activation of UPR
Sunirmal Paira and Biswadip Das
10 Assays to study IRE1 activation and signaling
Paloma Moraga, Raul Aravena, Hery Urra, and Claudio Hetz
11 Analysis of XBP1 contribution to hyperosmolarity-induced lipid synthesis.
Cecilia I. Casali, Leandro Parra, Luciana C. Erjavec, and María del Carmen Fernández Tome
12 Integrating an ER stress reporter for monitoring genome wide UPR-ER in budding yeast
Shuvadeep Maity, Asmita Ghosh, and Kausik Chakraborty
Part IV UPR studies in mammalian models
13 Analyze mouse knockout models of UPR pathway elements
Chao Zheng, Cheng Wang, Qiang Jie, and Liu Yang
14 In vitro stimulation of IRE1a/XBP1-deficient B cells with LPS
Huabin Zhu, Chen Jiang, Randal J. Kaufman, Honglin Li, and Nagendra Singh
15 Detection of PERK signaling in the central nervous system
Zhixin Lei, Sarrabeth Stone, and Wensheng Lin
Part V UPR in alternative models
16 Methods to study the mitochondrial unfolded protein response (UPRmt) in Caenorhabditis elegans
Simon Haeussler and Barbara Conradt
17 Drosophila Unfolded Protein Response (UPR) assays in vitro and in vivo
Hidetaka Kato, Deepika Vasudevan, and Hyung Don Ryoo
18 Protein preparation for proteomic analysis of the unfolded protein response in Arabidopsis thaliana
Yunting Pu and Federica Brandizzi
Part VI UPR and disease
19 Structure-based drug discovery of IRE1 modulators
Diana Pelizzari-Raymundo, Leif A. Eriksson, Eric Chevet, and Xavier Guillory
20 Chemical screening to detect ER stress regulators
Mehrnoosh Ghafouri, Chester B. Gauss, and Andrew M. Fribley
Includes cutting-edge methods and protocols
Provides step-by-step detail essential for reproducible result
Contains key notes and implementation advice from the experts
