Chemical genetic dissection of efferent IRE1α signalling

The Endoplasmic Reticulum is the cellular organelle primarily responsible for producing proteins on the secretory pathway, a pathway important in the production of biopharmaceuticals. One of the requirements for the successful production of a functional protein is correct folding of the polypeptide...

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Main Author: Sutcliffe, Louise Kathleen
Published: Durham University 2012
Subjects:
572
Online Access:http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.561006
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spelling ndltd-bl.uk-oai-ethos.bl.uk-5610062018-04-04T03:22:29ZChemical genetic dissection of efferent IRE1α signallingSutcliffe, Louise Kathleen2012The Endoplasmic Reticulum is the cellular organelle primarily responsible for producing proteins on the secretory pathway, a pathway important in the production of biopharmaceuticals. One of the requirements for the successful production of a functional protein is correct folding of the polypeptide sequence. During conditions such as viral infection, mutant protein expression and cell differentiation the endoplasmic reticulum is placed under conditions of stress. IRE1 is a protein kinase and endoribonuclease, which along with PERK and ATF6, forms part of the Unfolded Protein Response, the system by which the cell deals with the stress caused by a high protein load. IRE1 is capable of increasing the protein folding capacity of the ER, by upregulating chaperone proteins and reducing the load by attenuating translation, (protective response). This action is mediated by splicing of the mRNA coding for the bZIP transcription factor XBP-1. IRE1 is also capable of causing apoptotic responses via TRAF2 (cell injuring response) resulting in the activation of JNK and NFκB. In this study, using site directed mutagenesis a panel of IRE1 mutants was produced and screened for alterations to the protective and cell injuring responses. Of these the D711A mutant was shown in mouse embryonic fibroblasts to retain endoribonuclease activity, and to display an attenuated cell injuring response. When this mutant was applied to an industrial CHO cell line it appeared to exhibit an increase in biopharmaceutical productivity over the wild type IRE1, indicating its potential for use in the biopharmaceutical cell lines.572Durham Universityhttp://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.561006http://etheses.dur.ac.uk/5943/Electronic Thesis or Dissertation
collection NDLTD
sources NDLTD
topic 572
spellingShingle 572
Sutcliffe, Louise Kathleen
Chemical genetic dissection of efferent IRE1α signalling
description The Endoplasmic Reticulum is the cellular organelle primarily responsible for producing proteins on the secretory pathway, a pathway important in the production of biopharmaceuticals. One of the requirements for the successful production of a functional protein is correct folding of the polypeptide sequence. During conditions such as viral infection, mutant protein expression and cell differentiation the endoplasmic reticulum is placed under conditions of stress. IRE1 is a protein kinase and endoribonuclease, which along with PERK and ATF6, forms part of the Unfolded Protein Response, the system by which the cell deals with the stress caused by a high protein load. IRE1 is capable of increasing the protein folding capacity of the ER, by upregulating chaperone proteins and reducing the load by attenuating translation, (protective response). This action is mediated by splicing of the mRNA coding for the bZIP transcription factor XBP-1. IRE1 is also capable of causing apoptotic responses via TRAF2 (cell injuring response) resulting in the activation of JNK and NFκB. In this study, using site directed mutagenesis a panel of IRE1 mutants was produced and screened for alterations to the protective and cell injuring responses. Of these the D711A mutant was shown in mouse embryonic fibroblasts to retain endoribonuclease activity, and to display an attenuated cell injuring response. When this mutant was applied to an industrial CHO cell line it appeared to exhibit an increase in biopharmaceutical productivity over the wild type IRE1, indicating its potential for use in the biopharmaceutical cell lines.
author Sutcliffe, Louise Kathleen
author_facet Sutcliffe, Louise Kathleen
author_sort Sutcliffe, Louise Kathleen
title Chemical genetic dissection of efferent IRE1α signalling
title_short Chemical genetic dissection of efferent IRE1α signalling
title_full Chemical genetic dissection of efferent IRE1α signalling
title_fullStr Chemical genetic dissection of efferent IRE1α signalling
title_full_unstemmed Chemical genetic dissection of efferent IRE1α signalling
title_sort chemical genetic dissection of efferent ire1α signalling
publisher Durham University
publishDate 2012
url http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.561006
work_keys_str_mv AT sutcliffelouisekathleen chemicalgeneticdissectionofefferentire1asignalling
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