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 many human diseases defective proteins are made that damage the cell, so-called protein misfolding. In response to this, our cells reduce the rate at which they make new proteins in order to allow correction or destruction of these abnormal components. When the defect is corrected, protein synthesis increases once again.

The reduction of protein synthesis requires a protein called PERK, while the recovery involves a protein called GADD34. We have previously shown how mutations in the GADD34 gene protect against kidney damage in an animal model of protein misfolding. This led us to hypothesise that by developing drugs either to block GADD34 function or activate PERK, we might be able to protect tissues in diseases where protein misfolding is important, such as diabetes and stroke.

To help us identify potential drug targets, we are now studying how GADD34 and PERK are controlled by other components of the cell. We are using two main approaches: First, by studying how GADD34 and PERK are regulated in cultured human and mouse cells; and second, using genetically modified fruit flies (Drosophila melanogaster) to allow rapid identification of genes involved in the this system.

Technical Summary

Protein mis-folding in the endoplasmic reticulum (ER) is central to the pathogenesis of many human diseases (Marciniak & Ron, 2006 Physiol. Rev. 86:1133). Increased protein flux through the ER (ER stress) activates mechanisms that balance protein-folding capacity with protein load. These are referred to as the unfolded protein response (UPR). The UPR combines inhibition of protein translation with a transcriptional increase of ER chaperones. Translational inhibition follows phosphorylation of eIF2 by the ER stress-sensing kinase PERK; subsequent up-regulation of GADD34, an eIF2 phosphatase, reverses this inhibition. My previous studies into ER stress-induced cell death revealed an unexpected link between GADD34-dependent recovery of protein synthesis and the causation of tissue damage (Marciniak et al., 2004 Genes Dev. 18:3066). This suggested that inhibition of GADD34 or augmenting PERK activity might protect against ER stress-induced cell death in human disease.

Aims: 1. To characterise the post-translational modulation of PERK and GADD34 in order to identify novel factors that regulate the UPR.
2. To use Drosophila models to identify pathways linking ER stress and cell death.

Methods: 1. I will use techniques that I have previously developed (Marciniak et al., 2006 J Cell Biol. 172: 201) to study the PERK-inactivating factors in cell lysates. In particular I will use specific phosphatase inhibitors to determine pharmacologically the class(es) of phosphatase involved. I will over-express and knockdown individual phosphatase catalytic subunits in cultured 293T cells and assess the effect on PERK phosphatase activity. The effects of cellular stresses on PERK-inactivating activity will be studied. Biochemical fractionation will be used to isolate for identification the PERK-inactivating factors involved. Using a highly sensitive assay I have developed to measure eIF2 phosphatase activity, I will study the effects of GADD34 phosphorylation on its activity, both by in vitro phosphorylation and dephosphorylation of recombinant GADD34/PP1 holoenzyme. The effect of candidate GADD34-interactors will similarly be assessed. 2. I have generated Drosophila models of PERK/GADD34 signalling. Flies expressing PERK in the eye show a dramatic atrophic eye phenotype that is dependent on PERK catalytic activity and is rescued by co-expression of GADD34. Using these flies I will test candidate PERK and GADD34-modifying genes. Also, by crossing these flies with an existing 3000 fly P-element library I will screen for novel PERK and GADD34-interacting genes. Pathways involved will be manipulated using genetic and pharmacological tools in order to dissect their involvement in cellular survival and death during protein aggregation within the ER.
 
Description Addenbrooke's Charity, Fellowship
Amount £60,000 (GBP)
Organisation Addenbrooke's Charitable Trust (ACT) 
Sector Charity/Non Profit
Country United Kingdom
Start 09/2008 
End 09/2009
 
Description BLF Phd Studentship
Amount £93,156 (GBP)
Organisation British Lung Foundation (BLF) 
Sector Charity/Non Profit
Country United Kingdom
Start 10/2011 
End 09/2014
 
Description BLF Project Grant
Amount £118,839 (GBP)
Organisation British Lung Foundation (BLF) 
Sector Charity/Non Profit
Country United Kingdom
Start 09/2010 
End 09/2012
 
Description Diabetes UK, PhD Studentship
Amount £90,000 (GBP)
Organisation Diabetes UK 
Sector Charity/Non Profit
Country United Kingdom
Start 09/2009 
End 09/2012
 
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 MRC Clinical Research Training Fellowship
Amount £193,232 (GBP)
Organisation Medical Research Council (MRC) 
Sector Public
Country United Kingdom
Start 09/2011 
End 08/2014
 
Description MRC Senior Clinical Research Fellowship
Amount £1,630,935 (GBP)
Organisation Medical Research Council (MRC) 
Sector Public
Country United Kingdom
Start 03/2012 
End 02/2017
 
Description Theranostic Agents for Photothermal Treatment and Photo-acoustic Imaging of Mesothelioma
Amount £200,973 (GBP)
Organisation British Lung Foundation (BLF) 
Sector Charity/Non Profit
Country United Kingdom
Start 10/2017 
End 09/2020
 
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 Mesothelioma tissue microarray 
Description we have generated a large tissue microarray of mesothelioma tissue from 130 individuals representing all histological subtypes. We have linked anonymised clinical data for all cases 
Type Of Material Technology assay or reagent 
Provided To Others? No  
Impact This tissue microarray is the second largest in the world for this form of cancer and has the most comprehensive linked clinical data. Although we have not yet provided it to other groups, we are planning to do so in the near future to labs that have made requests for specific projects. 
 
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 Drosophila cell cycle checkpoints 
Organisation New York University
Department Department of Cell Biology
Country United States 
Sector Academic/University 
PI Contribution We generated a Drosophila model to study the effects of the kinase PERK on tissue growth. This was used to perform a genetic screen that identified grapes/CHK1 as mediating some of these effects.
Collaborator Contribution The collaborator's lab performed cell cycle staining of Drosophila eye imaginal discs and trained my PhD student in these techniques, which are now routinely used in my lab
Impact Malzer E., Daly M.L., Moloney A., Sendall T.J., Thomas, S.L. Ryder E., Ryoo H.D., Crowther D.C., Lomas, D.A. & Marciniak S.J. (2010). Impaired tissue growth is mediated by checkpoint kinase 1 (CHK1) in the integrated stress response. J. Cell Sci. 123: 2892-2900.
Start Year 2009
 
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 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