Backgrounder: How do we kill rogue cells?

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A molecular assassin literally punches its way into rogue cells say a team of Melbourne and London researchers. Their discovery is published today in Nature.

More effective treatments for cancer and viral diseases; better therapy for autoimmune conditions; a deeper understanding of the body’s defences enabling the development of more tightly focused immunosuppressive drugs—these are some of the wide-ranging possibilities arising from research published in the science journal Nature on Monday 31 October by research groups at Monash University and the Peter MacCallum Cancer Centre in Melbourne, and Birkbeck College in London.

Researchers at the three institutions have collaborated to unravel the molecular structure and function of the protein perforin, used by the body as a front-line weapon against viral infection.

The focus of the work can now switch to developing inhibitors and enhancers to regulate the molecule to medical advantage against cancer and other conditions such as cerebral malaria and juvenile diabetes.

Perforin is typically used by the T-cells and natural killer cells of the immune system to punch holes in the outer membranes of cells invaded by viruses. The immune system then uses these pores as access points for packages of toxic enzymes, or granzymes, which kill off the infected cells. The process prevents the replication and spread of virus particles within the body.

“This is a weapon of cleansing and death,” says Prof James Whisstock of Biochemistry and Molecular Biology at Monash University. “It’s the body’s key tool for killing off rogue cells.”

How these immune pore-forming proteins function has been a key question since the discovery of “haemolytic complement proteins” in the 1890s by the Nobel laureate Jules Bordet.

The new research has confirmed that the important parts of the perforin molecule are quite similar to those toxins deployed by bacteria such as anthrax, listeria and streptococcus. “The molecular structure of the warhead has been remarkably conserved throughout evolution. It has survived for close to two billion years, we think,” says Prof Joe Trapani, head of the Cancer Immunology Program at Peter Mac. “At some stage it seems that higher organisms may have captured that molecule from the enemy and are now using it back against them. So it’s both an offensive and defensive weapon.”

Trapani has been studying the mechanism of the immune system’s killer cells since 1985. “Until now,” he says, “perforin has been a real black box. No-one has really known how it all fits together to form a pore. And that’s really the point of this paper. That’s what we’ve cracked.”

He and National Health and Medical Research Council (NHMRC) fellow Dr Ilia Voskoboinik set the stage for the research around nine years ago by developing a means of producing and purifying sufficient perforin to allow the other two groups to probe the structure. But it wasn’t easy.

“This material is very toxic. So working out ways of producing it in cells in the lab without killing the cells was a major, major problem. Furthermore, perforin is extremely difficult to concentrate, and so the Peter Mac team had to work out how to develop forms of perforin suitable for the crystallography experiments.”

With access to a reliable supply of perforin, the other two groups set to work. Prof Helen Saibil and postdoctoral fellow Dr Natalya Lukoyanova of the Department of Crystallography at Birkbeck College used cryo-electron microscopy—where specimens can be observed in a natural state at very low temperature without using stains—to visualise rings of perforin molecules inserted in membranes. These rings have high walls and a large central hole, which is where the perforin punches through the cell membrane.

Meanwhile, Dr Ruby Law, a research fellow with Dr Whisstock was working out how to grow crystals of perforin.  When she succeeded, the team then collaborated with Dr Tom Caradoc-Davies from the nearby Australian Synchrotron and made extensive use of the micro crystallography beamline to finally reveal the complete molecular structure of perforin “This work is a dramatic illustration of the importance of the synchrotron,” says Whisstock. “We simply couldn’t have done it without this wonderful facility.”

The researchers knew from previous work that part of perforin (the MACPF domain) was similar to that found in bacterial toxins called cholesterol-dependent cytolysins (CDCs). The Saibil group had previously studied CDCs by electron microscopy, and they found that the molecules joined up into rings and then changed shape in a dramatic collapse to extrude coiled up regions of the protein into extended strands which penetrate the membrane to form a hole.

But when they compared the molecular structure of perforin with the cryo-electron micrograph, and then looked at the CDCs, the London group were puzzled to find that the MACPF domain in perforin was arranged inside out compared with the domain in bacteria. However, the Australians had noted another puzzling difference—a twist in the CDC molecules caused the top part of the structure to flip over relative to the region that contacts the membrane surface. This was a Eureka moment—the two observations fitted together to explain the perforin assembly. Remarkably, the same basic pore-forming machine, used by the immune system for defence and by bacteria for attack, works in opposite orientations in these two systems.

The weapon of death is a powerful molecule. Any functional aberrations in the perforin pathway can lead to dire medical consequences. It is thus tightly regulated by the body. But it is also an essential component of the immune system.

Because without perforin the immune system cannot kill. This means that mutations in the gene for perforin which result in a non-functional form are invariably fatal. They lead to a rare genetic disease known as familial hemophagocytic lymphohistiocytosis (FHL) type 2 in which the body cannot fight off infection.

As an important member of the body’s assassination team, however, another set of difficulties arise if perforin is merely deficient. Although the idea is controversial, many researchers regard the immune system as being involved in cancer surveillance, as proposed by Australian Nobel Prize laureate Sir Frank Macfarlane Burnett in the early days of immunology. In this role, perforin acts to wipe out precancerous cells before they become a problem. And there is evidence from mouse studies, says Trapani, that defective perforin leads to an upsurge in malignancy, particularly leukaemia.

Perforin is also the culprit when the wrong cells are marked for elimination, either in autoimmune disease conditions, such as early onset or type 1 diabetes, where the body attacks its own pancreatic cells, or in rejection, such as in bone marrow transplantation, where the immune system sets about ridding the body of “foreign” tissue.

So the development of a booster to counter deficient perforin could lead to more effective cancer protection, or therapy for acute diseases such as cerebral malaria, a nasty form of malaria which kills more than one million children a year in the developing world. And perforin inhibitors also could be used against conditions like diabetes or to provide better targeted immunosuppression to counter tissue rejection.

Candidate inhibitors have already been developed and, with a grant of more than $1 million from the Wellcome Trust, the collaborators have begun working with researchers at the University of Auckland to study their effects.

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