THE ROLE OF ENDOPLASMIC RETICULUM PROTEIN MISFOLDING IN CELL DEATH AND DISEASE
Lead Research Organisation:
University of Cambridge
Department Name: Cambridge Institute for Medical Research
Abstract
In order to grow, cells contain specialised structures called organelles. One of these is called the endoplasmic reticulum, which produces proteins. Cells suffer ER-stress when they cannot make secreted proteins normally and this can hinder their growth and even cause them to die. In many human diseases, such as diabetes and stroke, ER-stress impairs tissue survival.
We intend to study ER-stress in cells, fruit flies and mice. We hope to understand how ER-stress affects cell growth and causes death. We previously showed that two proteins, PERK and GADD34, play important roles in these phenomena. We now wish to understand how PERK and GADD34 are regulated. We wish also to identify new proteins involved in the response to ER-stress that alter cell growth.
Armed with this information, we will attempt to improve tissue survival in models of human disease. We are able to cause tissue damage in fruit flies or mice by genetically modifying them to make proteins that are mutated in human diseases. We will manipulate ER-stress signaling in these models and determine which are the proteins most likely to be useful drug targets in human disease.
We intend to study ER-stress in cells, fruit flies and mice. We hope to understand how ER-stress affects cell growth and causes death. We previously showed that two proteins, PERK and GADD34, play important roles in these phenomena. We now wish to understand how PERK and GADD34 are regulated. We wish also to identify new proteins involved in the response to ER-stress that alter cell growth.
Armed with this information, we will attempt to improve tissue survival in models of human disease. We are able to cause tissue damage in fruit flies or mice by genetically modifying them to make proteins that are mutated in human diseases. We will manipulate ER-stress signaling in these models and determine which are the proteins most likely to be useful drug targets in human disease.
Technical Summary
Aims: To understand how endoplasmic reticulum (ER) dysfunction affects tissue growth in disease and how its manipulation might provide novel therapies.
1. How is the PERK/GADD34 axis modulated during ER stress?
2. How are ER stress and cell growth signals integrated?
3. Can modulation of ER dysfunction promote the maintenance of secretory tissues in disease?
Methods: 1. We will identify GADD34‘s phosphorylated residues by mass spectroscopy and mutate these to determine their regulatory function. Using reagents we have generated (including GFP-GADD34 inducible cell lines, transgenic Drosophila) we will determine how phosphorylation affects GADD34 localisation, protein interactions, phosphatase activity and stability. 2. We will determine how PERK mediates CHK1 activation. We will induce ER stress or activate PERK directly using reagents we have described in cells (Marciniak et al., 2006 J Cell Biol. 172: 201) and flies (Malzer et al., 2010 J Cell Sci. 123: 2892) and measure DNA damage, CHK1 activity and CHK1-directed phosphatase activity. We have shown that PERK activation is insufficient for G1 cell-cycle arrest during ER stress (Malzer et al., 2010) and plan to identify the additional signals necessary. We will induce ER stress in cells deficient in UPR components and measure cell-cycle indices during ER-stress. We observe enhanced ER-stress-induced G2 arrest in p53-/- cells (unpublished) and so will determine how p53 modifies this. Using cell and fly reagents we have developed (Davies et al., 2009 JBC 284: 18202; Kroeger et al., 2009 JBC 284: 22793) we will determine the consequences of ER accumulation of ordered and disordered protein aggregates in order to dissect the pathways linking ER distension to NFkB activation. 3. In disease, ER dysfunction can manifest as ER-stress or ER-overload leading to loss of secretory tissue. We will use cell, mouse and fly models of (i) diabetes (ER-stress) and (ii) 1-antitrypsin deficiency (ER-overload) to determine how manipulation of these pathways can be employed for clinical benefit. (i) We will use genetic and pharmacological means to determine if manipulation of GADD34 toxicity can promote tissue survival in cell and mouse models of diabetes. We will determine how manipulation of CHK1 and p53 signalling affects the survival of ER-stress in these models. (ii) We will manipulate NF B signalling during ER-overload in cell and fly models of serpinopathies to determine its role in maintaining secretory tissue mass.
Opportunities: To identify novel targets for therapeutic intervention when ER dysfunction impairs tissue survival.
1. How is the PERK/GADD34 axis modulated during ER stress?
2. How are ER stress and cell growth signals integrated?
3. Can modulation of ER dysfunction promote the maintenance of secretory tissues in disease?
Methods: 1. We will identify GADD34‘s phosphorylated residues by mass spectroscopy and mutate these to determine their regulatory function. Using reagents we have generated (including GFP-GADD34 inducible cell lines, transgenic Drosophila) we will determine how phosphorylation affects GADD34 localisation, protein interactions, phosphatase activity and stability. 2. We will determine how PERK mediates CHK1 activation. We will induce ER stress or activate PERK directly using reagents we have described in cells (Marciniak et al., 2006 J Cell Biol. 172: 201) and flies (Malzer et al., 2010 J Cell Sci. 123: 2892) and measure DNA damage, CHK1 activity and CHK1-directed phosphatase activity. We have shown that PERK activation is insufficient for G1 cell-cycle arrest during ER stress (Malzer et al., 2010) and plan to identify the additional signals necessary. We will induce ER stress in cells deficient in UPR components and measure cell-cycle indices during ER-stress. We observe enhanced ER-stress-induced G2 arrest in p53-/- cells (unpublished) and so will determine how p53 modifies this. Using cell and fly reagents we have developed (Davies et al., 2009 JBC 284: 18202; Kroeger et al., 2009 JBC 284: 22793) we will determine the consequences of ER accumulation of ordered and disordered protein aggregates in order to dissect the pathways linking ER distension to NFkB activation. 3. In disease, ER dysfunction can manifest as ER-stress or ER-overload leading to loss of secretory tissue. We will use cell, mouse and fly models of (i) diabetes (ER-stress) and (ii) 1-antitrypsin deficiency (ER-overload) to determine how manipulation of these pathways can be employed for clinical benefit. (i) We will use genetic and pharmacological means to determine if manipulation of GADD34 toxicity can promote tissue survival in cell and mouse models of diabetes. We will determine how manipulation of CHK1 and p53 signalling affects the survival of ER-stress in these models. (ii) We will manipulate NF B signalling during ER-overload in cell and fly models of serpinopathies to determine its role in maintaining secretory tissue mass.
Opportunities: To identify novel targets for therapeutic intervention when ER dysfunction impairs tissue survival.
