Perforin-like proteins in health and disease

Project Overview

Figure 1: Left: pore formation by perforin-like proteins (PLPs) is typically thought to occur via ...

Figure 2: Left: a schematic phylogeny of mambrane-attack complex-perforin/cholesterol-dependent ...

Figure 3: Top: structure of the endoluminal domain of astrotactin-2 and, below, a schematic of the ...

We work on perforins, perforin-like proteins (PLPs) and the related bacterial cholesterol-dependent cytolysins (CDCs) [Gilbert et al., 2014; Ni and Gilbert, 2017]. These are mostly pore-forming proteins which target membranes either to allow the entry or exit of pathogens, or mechanisms of immune defence (Figure 1). However, some members of this membrane attack complex-perforin (MACPF)/CDC family have also been adapted to enable quite different processes, such as neural cell mechanisms of migration [Ni et al., 2016] (Figure 2).

There are three particular focuses of current work on MACPF/CDCs: (i) PLPs in Apicomplexan parasites like Plasmodium (PPLPs) and Toxoplasma (TgPLPs) and essential for infection and disease; (ii) mammalian perforin-2; (iii) proteins controlling neurodevelopmental processes.

(i) We have solved the structure of part of PPLP1 and are working on that of PPLP2; we are collaborating with a vaccine research group in London on the application of PPLP2. The next challenge is to understand the mechanism of the PPLPs and their pore structure. We have already solved the structure of TgPLP1, as a monomer and as an oligomer – a paper describing this work has been submitted.

(ii) Mammalian perforin-2 was only recently discovered, by a group in Miami with whom we collaborate [Podack and Munson, 2017]. We solved the structure of part of perforin-2 already but need to solve its pore structure and understand its activation. Perforin-2 is the essential frontline defence in humans against intracellular bacteria and differs in important ways from perforin-1 (e.g. it is a transmembrane protein).

(iii) We solved a structure of the soluble region of astrotactin-2, which guides cerebellar granule neurons along glial cells and is implicated in a host of neurodevelopmental diseases and early-onset Alzheimer’s [Ni et al., 2016] (Figure 3). We are now working on the structure of the full-length protein, as well as towards an improved understanding of its role using live cell imaging.

Training Opportunities

Structural methods: X-ray crystallography, cryo-EM and 3D reconstruction, SAXS

Biophysical methods: interaction analysis (SPR, AUC, ITC, DSC, DSF), pore-forming assays

Protein expression and purification: we use E. coli, mammalian and insect cell culture.

Theme

Protein Science & Structural Biology and Immunology & Infectious Disease

Admissions

Project reference number: 640

Funding and admissions information

Supervisors

Name Department Institution Country Email
Professor Robert Gilbert Structural Biology Oxford University, Henry Wellcome Building of Genomic Medicine GBR gilbert@strubi.ox.ac.uk

Gilbert RJ, Dalla Serra M, Froelich CJ, Wallace MI, Anderluh G. 2014. Membrane pore formation at protein-lipid interfaces. Trends Biochem. Sci., 39 (11), pp. 510-6. Read abstract | Read more

Pore-forming proteins (PFPs) interact with lipid bilayers to compromise membrane integrity. Many PFPs function by inserting a ring of oligomerized subunits into the bilayer to form a protein-lined hydrophilic channel. However, mounting evidence suggests that PFPs can also generate 'proteolipidic' pores by contributing to the fusion of inner and outer bilayer leaflets to form a toroidal structure. We discuss here toroidal pore formation by peptides including melittin, protegrin, and Alzheimer's Aβ1-41, as well as by PFPs from several evolutionarily unrelated families: the colicin/Bcl-2 grouping including the pro-apoptotic protein Bax, actinoporins derived from sea anemones, and the membrane attack complex-perforin/cholesterol dependent cytolysin (MACPF/CDC) set of proteins. We also explore how the structure and biological role of toroidal pores might be investigated further. Hide abstract

Ni T, Gilbert RJC. 2017. Repurposing a pore: highly conserved perforin-like proteins with alternative mechanisms. Philos. Trans. R. Soc. Lond., B, Biol. Sci., 372 (1726), Read abstract | Read more

Pore-forming proteins play critical roles in pathogenic attack and immunological defence. The membrane attack complex/perforin (MACPF) group of homologues represents, with cholesterol-dependent cytolysins, the largest family of such proteins. In this review, we begin by describing briefly the structure of MACPF proteins, outlining their common mechanism of pore formation. We subsequently discuss some examples of MACPF proteins likely implicated in pore formation or other membrane-remodelling processes. Finally, we focus on astrotactin and bone morphogenetic protein and retinoic acid-induced neural-specific proteins, highly conserved MACPF family members involved in developmental processes, which have not been well studied to date or observed to form a pore-and which data suggest may act by alternative mechanisms.This article is part of the themed issue 'Membrane pores: from structure and assembly, to medicine and technology'. Hide abstract

Ni T, Harlos K, Gilbert R. 2016. Structure of astrotactin-2: a conserved vertebrate-specific and perforin-like membrane protein involved in neuronal development. Open Biol, 6 (5), Read abstract | Read more

The vertebrate-specific proteins astrotactin-1 and 2 (ASTN-1 and ASTN-2) are integral membrane perforin-like proteins known to play critical roles in neurodevelopment, while ASTN-2 has been linked to the planar cell polarity pathway in hair cells. Genetic variations associated with them are linked to a variety of neurodevelopmental disorders and other neurological pathologies, including an advanced onset of Alzheimer's disease. Here we present the structure of the majority endosomal region of ASTN-2, showing it to consist of a unique combination of polypeptide folds: a perforin-like domain, a minimal epidermal growth factor-like module, a unique form of fibronectin type III domain and an annexin-like domain. The perforin-like domain differs from that of other members of the membrane attack complex-perforin (MACPF) protein family in ways that suggest ASTN-2 does not form pores. Structural and biophysical data show that ASTN-2 (but not ASTN-1) binds inositol triphosphates, suggesting a mechanism for membrane recognition or secondary messenger regulation of its activity. The annexin-like domain is closest in fold to repeat three of human annexin V and similarly binds calcium, and yet shares no sequence homology with it. Overall, our structure provides the first atomic-resolution description of a MACPF protein involved in development, while highlighting distinctive features of ASTN-2 responsible for its activity. Hide abstract

Podack ER, Munson GP. 2016. Killing of Microbes and Cancer by the Immune System with Three Mammalian Pore-Forming Killer Proteins. Front Immunol, 7 pp. 464. Read abstract | Read more

Immunology is the science of biological warfare between the defenses of our immune systems and offensive pathogenic microbes and cancers. Over the course of his scientific career, Eckhard R. Podack made several seminal discoveries that elucidated key aspects of this warfare at a molecular level. When Eckhard joined the complement laboratory of Müller-Eberhard in 1974, he was fascinated by two questions: (1) what is the molecular mechanism by which complement kills invasive bacteria? and (2) which one of the complement components is the killer molecule? Eckhard's quest to answer these questions would lead to the discovery C9 and later, two additional pore-forming killer molecules of the immune system. Here is a brief account of how he discovered poly-C9, the pore-forming protein of complement in blood and interstitial fluids: Perforin-1, expressed by natural killer cells and cytotoxic T lymphocytes; and Perforin-2 (MPEG1), expressed by all cell types examined to date. All the three killing systems are crucial for our survival and health. Hide abstract