Dynamics and pathways of assembly in membrane pore formation

Lead Research Organisation: University College London
Department Name: London Centre for Nanotechnology

Abstract

Pore-forming proteins are crucial armaments in the continuous battle between living organisms and the pathogens that threaten their fitness and survival. These proteins act on cells, which are the micrometre-scaled, basic units of all forms of life. Cells are separated and protected from their environment by a thin membrane. Pathogens such as bacteria can release pore-forming proteins ("toxins") that drill holes in the membranes of healthy cells in the host organism, to release nutrients for the bacteria, to invade these cells and/or kill them. Patients affected by bacterial pneumonia, for example, suffer from the devastating effects of such a toxin, pneumolysin, on lung tissue. The immune system, however, uses a similar mechanism to kill germs and infected or cancerous cells, thus preventing them from doing further damage to the organism. It secretes related, but somewhat different pore-forming proteins to perforate the membranes of such unwanted invaders.

To perform these tasks, pore-forming proteins have developed sophisticated drilling mechanism. These proteins can convert from a soluble form in the aqueous, cellular environment into a very different form, in which 20-50 protein molecules assemble into a ring-shaped pore bound to the membrane. We can look at these forms with X-rays or electrons to deduce their three-dimensional structures. Thanks to such experiments, we now have a reasonably clear picture of the soluble proteins and their pore structure in the membrane. For some pore-forming proteins, scientists have even identified the changes inside the proteins which make this transition possible.

However, if we wish to design drugs that prevent such pores from being formed, as in the example of bacterial pneumonia indicated above, it would be useful to know more about the steps in their formation. It is exactly this pore assembly that is still largely enigmatic. In this project, we will try to answer some specific questions about membrane pore formation. We would like to know how the proteins assemble on the membrane. Do they assemble one by one, or do they first form larger units that subsequently assemble in a pore? Do the proteins first need to assemble on the membrane, or can they dock in the membrane and assemble in pores afterwards? And at what point in this process will the membrane that is surrounded by the assembled protein be extruded to create a hole?

To investigate the dynamics of this process, we rely on a technique called atomic force microscopy. Atomic force microscopy is the small-scale equivalent of reading Braille: With a tiny artificial finger, we feel the pore-forming proteins while they assemble on the membrane. Whereas X-ray crystallography and electron microscopy are limited to static samples, atomic force microscopy can probe active proteins while they are at work. We will thus apply atomic force microscopy to the membranes that are being exposed to attack by pore-forming proteins. Meanwhile, we will benefit from the more detailed views provided by electron microscopy to identify intermediate assemblies of pore-forming proteins, that are trapped by chemical bonds or by lowering the temperature. Electron microscopy will thus provide highly detailed pictures of pore forming proteins in different states of assembly and atomic force microscopy will enable us to see how the proteins transit between these different states.

Technical Summary

We will use real-time atomic force microscopy (AFM), single particle electron microscopy (EM) and electron tomography to track the dynamics and the pathways of assembly of pore-forming proteins on and in membranes. We will focus on the superfamily of cholesterol dependent cytolysins (CDCs) and membrane attack complex/perforin (MACPF) pore-forming proteins. Though atomic structures of CDC and MACPF proteins have been determined for the soluble monomers, along with low resolution maps of the membrane-inserted pores, the dynamics and pathways of assembly are much harder to address experimentally. To find assembly pathways, we will trap intermediate assemblies with disulphide locked variants and reduced temperatures, and determine their structures by EM. Moreover, benefitting from recent developments on fast-scanning AFM, we will follow the assembly and pore formation by real-time imaging of the functional proteins on planar-supported model membranes. Combining the two methods, we will determine the sequential steps in the assembly of the prepore and pore state of bacterial CDCs and of the MACPF protein perforin. By probing inside the rings with AFM, we will also investigate the fate of the lipid bilayer in the pores upon assembly of bacterial CDCs. We will elucidate biophysical mechanisms that are fundamental to pathogen attack and immune defence. This information is anticipated to create opportunities for new drug design, either by stabilising pore-forming proteins to prevent pore formation, or in stabilising or enhancing their membrane-bound forms, in case of deficient pore-forming activity.

Planned Impact

This proposal aims at elucidating the biological and biophysical mechanisms of membrane pore formation by bacterial toxins and immune mediators. The superfamily of membrane attack complex/perforin (MACPF) and cholesterol-dependent cytolysin (CDC) proteins that it focuses on are of significant medical importance. The CDC perfringolysin O rapidly induces irreversible cellular injury in a deadly form of gangrene that is caused by the bacterium Clostridium perfringens. The CDC pneumolysin is a major virulence factor of Streptococcus pneumoniae, at the root of bacterial pneumonia, still a major cause of death and illness throughout the world despite the widespread use of antibiotics. When released in the lungs, pneumolysin damages the lung tissue and its blood vessels. Antibiotics may exacerbate lung damage because they are designed to kill the bacteria by breaking them open, which leads to the further release of pneumolysin. Perforin is the main weapon of natural killer cells. It punches holes in virally infected or cancerous cells that have been detected by the immune system, and delivers lethal granzymes through these holes. Babies born with defective perforin succumb to viral infections or tumours early in life. On the other hand, if perforin is too active, normal cells can be incorrectly killed.

By elucidating the mechanism of membrane pore formation by pore-forming proteins, we will create new opportunities for drug design: e.g., the prevention of pore formation by pneumolysin would be a significant advance in the treatment of bacterial pneumonia; the ability to control the activity of MACPF proteins in the human immune system could be an important means of regulating the immune response during and after tissue and organ transplantation or could alleviate the perforin-dependent cytotoxicity in autoimmune diabetes.

In addition, the proposed methodology for studying membrane pore formation is not restricted to pore-forming proteins alone. It can be applied to a variety of other medically relevant interactions between membranes and macromolecules. Examples of this are antimicrobial peptides that are currently investigated as new therapies against bacterial infections, as well as pH-sensitive polymers that are used for intracellular drug (e.g., gene and RNA therapies) delivery across the barrier formed by endosomal membranes after cellular drug in-take via endocytosis. We anticipate that the development of such novel therapeutic approaches will be enhanced by molecular-scale understanding as can be achieved with the methods outlined in this proposal.

On the shorter term (several years), this research will thus benefit pharmaceutical industry and biomedical SMEs. On the longer term (10-20 years, given the lead times for drug development), it will have an impact on healthcare practitioners and patients.

Further impact can be achieved on the AFM technology. With this project, we propose a new application of fast-scanning AFM techniques that have only recently been developed. For such a young technology, feedback based on relevant applications is crucial. It helps to adjust the technology such that it can make a larger impact over a wider range of applications, which will benefit nanotechnological SMEs.

Given the power of high-quality images and movies such as can be produced by in particular AFM, we also anticipate an educational impact to the lay audience by demonstrating a powerful and biomedically relevant application of basic sciences and nanotechnology.

Publications

10 25 50
 
Title Film The Demiurge inspired by our research 
Description Inspired by our research on imaging DNA by high-resolution atomic force microscopy, British artists AL and AL made a ski-fi artistic film, named The Demiurge, which has its world premiere February 2016 in a widely attended artistic exhibition in Manchester, and which was co-funded by the Wellcome Trust. 
Type Of Art Film/Video/Animation 
Year Produced 2016 
Impact Over 4000 visitors in first week of exhibition. 
URL http://homemcr.org/exhibition/al-al-incidents-of-travel-in-the-multiverse/
 
Description We have viewed bacterial nanodrills in action. Briefly, bacteria can gain access to food held in a healthy cell by perforating its protective membrane. We have imaged the nanodrills that do this peroration and gained insights into their way of operation. This has been expanded to pore forming proteins in the immune system, helping us to understand mechanisms behind certain immune diseases.
Exploitation Route The obtained understanding can be used to optimise drug development programmes against, e.g., bacterial pneumonia.
Sectors Healthcare,Pharmaceuticals and Medical Biotechnology

URL https://www.ucl.ac.uk/mathematical-physical-sciences/maps-news-publication/maps1434
 
Description Methodology developed in this grant has been translated to experiments on novel antimicrobial peptides developed at NPL, and helped to elucidate their functional mechanisms as antibiotics.
First Year Of Impact 2013
Sector Pharmaceuticals and Medical Biotechnology
Impact Types Societal

 
Description BBRSC CASE PhD studentship
Amount £102,000 (GBP)
Funding ID BB/M503113/1 
Organisation Biotechnology and Biological Sciences Research Council (BBSRC) 
Sector Public
Country United Kingdom
Start 10/2014 
End 09/2018
 
Description BBSRC ALERT
Amount £173,000 (GBP)
Funding ID BB/R000042/1 
Organisation Biotechnology and Biological Sciences Research Council (BBSRC) 
Sector Public
Country United Kingdom
Start 08/2017 
End 08/2018
 
Description EPSRC Equipment Funding
Amount £711,385 (GBP)
Funding ID EP/M028100/1 
Organisation Engineering and Physical Sciences Research Council (EPSRC) 
Sector Academic/University
Country United Kingdom
Start 04/2015 
End 03/2016
 
Description Impact Acceleration Account
Amount £10,000 (GBP)
Funding ID BB/IAA/UCL/15 
Organisation Biotechnology and Biological Sciences Research Council (BBSRC) 
Sector Public
Country United Kingdom
Start 07/2015 
End 09/2016
 
Description MRC Research Grant
Amount £442,227 (GBP)
Funding ID MR/R000328/1 
Organisation Medical Research Council (MRC) 
Sector Academic/University
Country United Kingdom
Start 07/2018 
End 06/2021
 
Description Responsive mode grant
Amount £458,449 (GBP)
Funding ID BB/N015487/1 
Organisation Biotechnology and Biological Sciences Research Council (BBSRC) 
Sector Public
Country United Kingdom
Start 10/2016 
End 10/2019
 
Title High-resolution atomic force microscopy 
Description Atomic force microscopy techniques to visualise DNA / nucleic acid / oligonucleotidedouble helix structure 
Type Of Material Technology assay or reagent 
Year Produced 2013 
Provided To Others? Yes  
Impact Related work has benefitted industrial research by MedImmune (branch of AstraZeneca) and Syngenta. 
 
Description AstraZeneca collaboration 
Organisation AstraZeneca
Country United Kingdom 
Sector Private 
PI Contribution Our partner is interested in DNA strand-break repair as is required for the proliferation of tumour cells, in particular when treated by e.g. radio-/chemo-therapy. They have an anticancer drug on the market that suppresses this repair process to the detriment of the tumour cells. We use single-molecule biophysics approaches, as developed refined in various research-council funded projects, to identify the mechanism by which this drug interferes with DNA strand-break repair, with the aim to provide our partner with a better scientific understanding, which can in turn facilitate further optimisation of their drug in terms of efficacy and selectivity for cancer cells.
Collaborator Contribution They provide the background knowledge on the biology and medical context for this research. They provide materials (purified proteins, inhibitor drugs) and expertise.
Impact The collaboration is in a too early stage (only few months on-going).
Start Year 2017
 
Description Imperial College London 
Organisation Imperial College London
Department Imperial College Trust
Country United Kingdom 
Sector Charity/Non Profit 
PI Contribution Based on our research on the biological nuclear pore complex, we have proposed mechanisms via which artificial nanopores may be designed as selective valves. With collaborators at Imperial College, we currently attempt to implement these ideas in a working system. On a different topic, our work on pore forming proteins has helped us to initiate a collaboration with structural biologists at Imperial College, where we attempt to use similar methodology to elucidate mechanism via which the immune system attacks bacteria. We have now also started to collaborate on the formation of quadruply stranded DNA structures, as targeted by potential anticancer agents.
Collaborator Contribution Nanopore devices. Purified proteins. DNA constructs. Expertise.
Impact This is a multi-disciplinary collaboration, involving physicists, chemists, engineers and biologist. It is still rather early to highlight particular outcomes.
Start Year 2014
 
Description Industrial collaboration with AFM manufacturer Bruker Nano 
Organisation Bruker Corporation
Department Bruker Nano
Country Germany 
Sector Private 
PI Contribution Following successful high-resolution atomic force microscopy (AFM) imaging, we have signed a joint development agreement with world-leading AFM manufacturer Bruker Nano (formerly Veeco, formerly Digital Instruments), on testing and developing protocols on prototype AFM equipment. Bruker Nano contributes in-kind to this project. Our work with their instrumentation and probes has provided Bruker with AFM images of the DNA double helix (and protocols for acquiring these), as well as an assessment of probe tip sharpness (both by benchmarking on DNA and on assemblies of pore forming proteins.
Collaborator Contribution Provision of and access to latest commercial AFM equipment (including beta-version pre market release), provision of AFM proves, all at zero or greatly reduced price.
Impact Multidisciplinary - involving engineering, physics and biology. Outcomes of broad and general use are protocols and instructions for double-helix-resolution imaging of DNA in liquid, see, e.g., a webinar on this (http://www.bruker.com/service/education-training/webinars/afm.html), which helps AFM manufacturer Bruker and its representation in the UK. Technical feedback from our side has helped Bruker to optimise its products before and after market release. Another outcome is the visualisation of membrane lesions and of prepore-state bacterial toxins diffusing on a membrane surface, elucidating the pathways of membrane pore formation by bacterial toxins, as well as understanding of mechanism by which antimicrobial peptides (potential next-generation antibiotics) attack bacteria.
Start Year 2012
 
Description MedImmune 
Organisation MedImmune
Country United States 
Sector Private 
PI Contribution Imaging of cell surface receptors, to help MedImmune (Cambridge) understand the mechanisms by which monoclonal antibody drugs target cell surface receptors.
Collaborator Contribution Technical advise, PhD student supervision, purified proteins, financial contributions (cash) to support research.
Impact The results are largely confidential, but include understanding of the working mechanisms of monoclonal antibody drugs targeting cell surface receptors. We have acquired single-molecule kinetic data of these drugs binding to their receptors embedded in membranes, where the idea is that such kinetic data inform further drug development.
Start Year 2014
 
Description National Physical Laboratory 
Organisation National Physical Laboratory
Country United Kingdom 
Sector Academic/University 
PI Contribution We have provided the technology to visualise how next-generation antibiotics (de-novo designed antimicrobial peptides) attack bacteria.
Collaborator Contribution Design of antimicrobial peptides. Expertise.
Impact Multidisciplinary, involving physicists, chemists, bioengineers, and biologists. This collaboration has helped to put forward various antimicrobial peptides as (potential) next-generation antibiotics.
Start Year 2010
 
Description Peter MacCallum Cancer Centre 
Organisation Peter MacCallum Cancer Centre
Country Australia 
Sector Hospitals 
PI Contribution Insights in the pathways of membrane pore formation by perforin, a component of the human immune system.
Collaborator Contribution Scientific advise and purified proteins.
Impact We have determined the self-assembly mechanism by which the immune protein perforin forms pores in cancerous and virus-infected cells in our bodies (published in Nature Nanotechnology). This has given an indication for explaining how patients with perforin deficiencies show enhanced susceptibility to blood cancer. In addition, on-going research has highlighted reasons why certain cancer cells may be more or less susceptible for immune clearance in latest cancer immunotherapies.
Start Year 2012
 
Description University of Leicester 
Organisation University of Leicester
Country United Kingdom 
Sector Academic/University 
PI Contribution Insights in mechanism of bacterial toxins that are subject of drug development programmes in Leicester
Collaborator Contribution Provision of purified proteins.
Impact Insights in pathways of membrane pore formation by the bacterial toxin suilysin (published in eLide).
Start Year 2012
 
Description Utrecht collaboration 
Organisation University Medical Center Utrecht (UMC)
Country Netherlands 
Sector Academic/University 
PI Contribution We carry out nanoscale characterisation of life bacteria as they are attacked by immune proteins (complement) in serum, with the aim to determine which are the mechanism by which the immune system clears our body from harmful bacteria.
Collaborator Contribution Scientific expertise, different strains of bacteria, purified proteins.
Impact We have identified a new role of certain immune enzymes in killing bacteria. This can now be used to guide immune therapies to bacterial infections.
Start Year 2016
 
Description Food industry talks 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Professional Practitioners
Results and Impact At Leatherhead Food Research and at Mars Corporation, talk on nanotechnology sparked questions about the use of it for food industry.

Follow-up emails.
Year(s) Of Engagement Activity 2012,2014
 
Description Lego4Nano workshop in Beijing 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Public/other audiences
Results and Impact My PhD students provided the expertise in atomic force microscopy that was essential in this UK-China workshop, sponsored by Lego, to build a low-cost microscope

the workshop was widely reported in the media, including Chinese national newspapers.
Year(s) Of Engagement Activity 2013,2014
URL http://www.london-nano.com/news-and-events/news/2014-lego2nano-launches
 
Description School talks 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach Regional
Primary Audience Schools
Results and Impact Our school talks typically sparked questions and discussions about nanotechnology, about physics and biology.

Follow-up emails.
Year(s) Of Engagement Activity 2009,2010,2011,2012,2013