Projects / Programmes
Molecular Description of Lipid Membrane Changes in Disease
| Code |
Science |
Field |
Subfield |
| 1.05.00 |
Natural sciences and mathematics |
Biochemistry and molecular biology |
|
| Code |
Science |
Field |
| P310 |
Natural sciences and mathematics |
Proteins, enzymology |
| Code |
Science |
Field |
| 1.06 |
Natural Sciences |
Biological sciences |
Proteins, Membranes, Immunity, Pathogenic Bacteria, MACPF/CDC protein family, Membrane Remodelling, Perforin, Listeriolysin, Ostreolysin
Researchers (21)
Organisations (4)
Abstract
Protein-membrane interactions have a central role in many fundamental biological processes and diseases, including bacterial infections and parasitism. Protein-induced damage of plasma or intracellular membranes integrity leads ultimately to cell death. It is, therefore, important to understand how proteins affect membranes at the molecular level. How proteins break the membrane barrier is still not satisfactorily explained for many diverse groups of membrane interacting proteins. Subtle mechanisms have evolved that allow the invasion of molecules and even organisms through cells without gross damage of membranes or nearby tissues. They involve dynamic remodelling of membranes, similar to those in movement, division, extension of neuronal arbors and vesicle trafficking. Here we plan to study some of such activities of an important protein superfamily, the membrane attack complex/perforin and cholesterol-dependent cytolysin (MACPF/CDC) proteins.
The MACPF/CDC protein family ()350 members) was defined on the basis of sequence similarity between proteins of the immune systems Membrane-Attack Complex (MAC) of the complement system and PerForiN (PFN), and structural similarity to cholesterol-dependent cytolysins (CDC). The similar architecture by analogy implies that MACPF proteins might act by a similar mechanism of transmembrane pore formation. MACPFs and CDCs thus represent an ancient protein lineage associated with fundamental processes in infection and immunity and, hence, they are collectively referred to as the MACPF/CDC superfamily. Transmembrane pore formation is the hallmark characteristic of this protein family, however, membrane perforation is not all that these proteins are capable of. It was shown that MACPF/CDCs may form incomplete rings or arcs on the surface of the membrane, which are functional and possibly lined with membrane lipids in the ion conductive pathway. Furthermore, not all of the MACPF/CDC proteins possess pore forming activity and it remains to be determined even if membrane interactions are needed for the activity of all of them. A range of different effects could be in play, including fusogenic properties as observed for some of the CDCs. We have recently observed that human PFN causes invaginations and/or intra-luminal vesicles (ILVs) in giant unilamellar vesicles (GUVs) (Supplementary material). The formation of such structures depended on functional PFN.
In the proposed project we plan to study biochemical and some structural properties of protein-lipid complexes induced by several MACPF/CDC proteins: human PFN, listeriolysin O (LLO) from Listeria monocytogenes, and a fungal bicomponent pore forming protein ostreolysin (Oly). While PFN and LLO are extensively biochemically characterised, properties and function of Oly and other similar proteins are largely unkown. The main aim of this project is to describe the molecular mechanism of large- and small-scale changes in membrane structure induced by these proteins. Large-scale changes in membrane geometry are introduced by protein binding to the one monolayer of the membrane as highlighted by preliminary data for PFN. This newly observed membrane remodelling by PFN may have implications for its biology and may open new horizons to understand how it kills target cells. Small-scale changes in membrane structures are expected in incomplete pores that are implicated in the pathogenic mechanism exerted by L. monocytogenes, the causative agent of listeriosis. Specific goals of this project are:
i) Characterisation of novel fungal bicomponent MACPF/CDCs,
ii) Development of probes for labelling of membranes,
iii) Determination of LLO atomic structure,
iv) Determination of structural and functional properties of MACPF/CDCs pores, and
v) Description of large-scale membrane remodelling induced by MACPF/CDC proteins.
Significance for science
The project focused on research of basic biomedical processes and important molecules: role of proteins in remodelling of cellular membranes, 3D structure of some pore-forming proteins and new group of MACPF proteins that specifically associate with membrane nanodomains ("lipid rafts"). Cellular membranes remodelling by proteins is essential in membrane-membrane interactions such as fusion and fission or single membrane reshaping. It is in common to known models of membrane remodelling studied so far that all mostly consider membrane integrity constant. We have described pore formation by some of the medically important proteins and characterise their functional properties. We contributed to understanding of interactions of proteins with lipid membranes. We have also described a novel group of bi-component MACPF/CDC proteins, which form pores and characterisation of their properties is important for understanding of their, yet unknown, biological activity. The low-molecular weight component is particularly interesting. We have shown that it binds to lipid rafts and that it can be used in cell-biology applications. Since their discovery lipid rafts attracted an enormous interest in scientific community. In this respect, development of fluorescent probes specific for particular components or clusters of molecules found in lipid rafts is necessity. The outcomes of the project are important also because they enable better understanding of medically important MACPF/CDC proteins. We have prepared an expression system for mouse perforin and define its interactions with lipid membranes. Successful preparation of mouse perforin and some of the model lipid systems that were prepared during the project will open doors to further experimentation, on cells and experimental animals, which will finally resolve this mechanism so important for the immune system. Also the understanding of pathogenicity of L. monocytogenes is medically extremely important. We succeeded in describing properties of pores formed by listeriolysin O.
Significance for the country
The applicative value of the proposed study for Republic of Slovenia is multiple: promotion of science and scientific discoveries in the field of protein-membrane interactions, training of young researchers, development of new technologies and methods, development of new research tools in lipid biochemistry, etc. The further development of modern biophysical approaches, such as surface plasmon resonance and thermophoresis, together with development of some of the model lipid systems, will render Slovenian science more competitive. The approaches are relatively new to science and not many groups explore it fully. The group of principal investigator is becoming very experienced in protein-lipid interaction studies by these approaches. This project will enable to develop the expertise even further by designing novel assays and thus extending capabilities of this interesting and useful method.
Most important scientific results
Annual report
2011,
2012,
2013,
final report,
complete report on dLib.si
Most important socioeconomically and culturally relevant results
Annual report
2011,
2013,
final report,
complete report on dLib.si
