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.

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 mipulation 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.
 
Description cited by Lord Alton of Liverpool (speech on Mesothelioma Bill 17 July 2013 - Hansard Column 751)
Geographic Reach National 
Policy Influence Type Citation in other policy documents
URL http://www.publications.parliament.uk/pa/ld201314/ldhansrd/text/130717-0001.htm
 
Description Alpha-1 Foundation Project grant
Amount $187,456 (USD)
Funding ID RG84241 
Organisation Alpha-1 Foundation 
Sector Charity/Non Profit
Country United States
Start 07/2016 
End 06/2018
 
Description CRUK Cancer Clinical Research Fellowship
Amount £203,919 (GBP)
Organisation Cancer Research UK 
Sector Charity/Non Profit
Country United Kingdom
Start 10/2013 
End 12/2014
 
Description Diabetes UK PhD studentship
Amount £95,496 (GBP)
Funding ID 12/0004595 
Organisation Diabetes UK 
Sector Charity/Non Profit
Country United Kingdom
Start 10/2013 
End 09/2016
 
Description EPSRC IRC in Targeted Delivery for Hard-to-Treat Cancers
Amount £10,275,035 (GBP)
Funding ID EP/S009000/1 
Organisation Engineering and Physical Sciences Research Council (EPSRC) 
Sector Public
Country United Kingdom
Start 10/2018 
End 09/2024
 
Description Wellcome Trust PhD studentship
Amount £90,000 (GBP)
Organisation University of Cambridge 
Department Cambridge Institute for Medical Research (CIMR)
Sector Academic/University
Country United Kingdom
Start 10/2013 
End 09/2016
 
Title FRET based endoplasmic reticulum crowding probe 
Description Developed FRET-based system to report molecular crowding within the endoplasmic reticulum 
Type Of Material Technology assay or reagent 
Year Produced 2018 
Provided To Others? Yes  
Impact Holcman D, Parutto P, Chambers JE, Fantham M, Young LJ, Marciniak SJ, Kaminski CF, Ron D, Avezov E. (2018). Single particle trajectories reveal active endoplasmic reticulum luminal flow. Nat Cell Biol 20:1118-1125 doi: 10.1038/s41556-018-0192-2. 
URL https://www.nature.com/articles/s41556-018-0192-2
 
Title ROVI (Rotor based organelle viscosity imaging) 
Description Microscopic viscosity (microviscosity) is a key determinant of diffusion in the cell and defines the rate of biological processes occurring at the nanoscale, including enzyme-driven metabolism and protein folding. Here we establish a rotor-based organelle viscosity imaging (ROVI) methodology that enables real-time quantitative mapping of cell microviscosity. This approach uses environment-sensitive dyes termed molecular rotors, covalently linked to genetically encoded probes to provide compartment-specific microviscosity measurements via fluorescence lifetime imaging. ROVI visualized spatial and temporal dynamics of microviscosity with suborganellar resolution, reporting on a microviscosity difference of nearly an order of magnitude between subcellular compartments. In the mitochondrial matrix, ROVI revealed several striking findings: a broad heterogeneity of microviscosity among individual mitochondria, unparalleled resilience to osmotic stress, and real-time changes in microviscosity during mitochondrial depolarization. These findings demonstrate the use of ROVI to explore the biophysical mechanisms underlying cell biological processes. 
Type Of Material Technology assay or reagent 
Year Produced 2018 
Provided To Others? Yes  
Impact First method to permit comparable microviscosity measurement using the same fluorescent probe 
URL https://pubs.acs.org/doi/10.1021/acsnano.8b00177
 
Description Epithelial and macrophage dysfunction in alpha1-antitryspin deficiency 
Organisation University of Cambridge
Department Cambridge Institute for Medical Research (CIMR)
Country United Kingdom 
Sector Academic/University 
PI Contribution I now co-supervise JD with Prof DL in which she studies the effects of antitrypsin mutants synthesised locally within airway epithelia - both Prof DL and I help design experiments and interpret their results
Collaborator Contribution New shared PhD Student - Dr JD (medical doctor)
Impact New cell lines generated and being used in this project
Start Year 2011
 
Description Mesothelioma and ER stress 
Organisation Papworth Hospital NHS Foundation Trust
Country United Kingdom 
Sector Public 
PI Contribution We envisaged and generated a tissue microarray with which to determine if endoplasmic reticulum stress pathways are activated in mesothelioma tissues
Collaborator Contribution Access to archived patient tissues used in generation of tissue microarray
Impact This collaboration involves clinicians (including myself), scientists (from my lab) and pathologists. We have generated a tissue microarray representing over 100 individual patients and all histological subtypes of the disease. This is to be linked with a comprehensive clinical database.
Start Year 2010
 
Description Surfactant C mutant models 
Organisation University of Pittsburgh
Department Division of Pulmonary, Allergy, and Critical Care Medicine
Country United States 
Sector Academic/University 
PI Contribution My group generated Drosophila model with which to study the in vivo effects of the accumulation of mutant surfactant proteins
Collaborator Contribution In collaboration with workers at U Penn, we have generated new fly models of surfactant protein aggregation. These are being used in ongoing projects. The Beers labs provided the original human mutant constructs and advice
Impact Model generated and currently being used - no publications yet
Start Year 2011
 
Description Cambridge Chemistry Challenge 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Schools
Results and Impact Approximately 50 pupils who had scored highly in the national Cambridge Chemistry Challenge attended for a series of lectures and practicals in the Chemistry Department, Cambridge. My lectured focused on medical aspects of chemistry and stimulated a lively Q&A session.

Too early to say, but previously this scheme has prompted attendees to apply successfully to study either Natural Sciences or Medical Sciences in Cambridge.
Year(s) Of Engagement Activity 2014
URL https://www.facebook.com/pages/Cambridge-Chemistry-Challenge-C3L6/150583718336215
 
Description Hosted College Student 4-week research project 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? Yes
Geographic Reach Local
Primary Audience Schools
Results and Impact A-level college students undertake a 4-week research project in my group, prepare a written research report and then present the findings in poster format at a meeting hosted by the Nuffield Foundation.

One student (from 2006) chose to continue in biomedical sciences and is currently a medical student at Oxford University. A second student (from 2008) is has been accepted to study medicine at Cambridge University. The third has a research technician post during his gap year - plans to apply to University to study natural sciences. The 2013 student is completing his A-levels and is applying to study medicine.
Year(s) Of Engagement Activity 2006,2008,2009,2010,2014
 
Description Sutton Trust Lecture (Cambridge) 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? Yes
Geographic Reach National
Primary Audience Schools
Results and Impact More than 50 students attended my scientific lecture. This was followed by a lively Q&A session. Subsequently, one student has maintained email contact for further research materials.

Too early to say.
Year(s) Of Engagement Activity 2014
URL http://www.study.cam.ac.uk/undergraduate/access/year12summerschool/