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Programmed cell death targets discovered

Apoptotic processes, i.e. cell death mediated by intracellular programmes, have been implicated in a variety of diseases. Apoptotic processes eliminate superfluous or irreparably damaged cells from the body; however, defective apoptotic processes harm the organism. New research results show that processes at the mitochondrial membrane might be excellent targets for pharmaceutical interference with apoptosis.

Prof. Dr. Ana J. García-Sáez, who is of Spanish origin, has held a professorship at the Interfaculty Institute of Biochemistry at the University of Tübingen (IFIB) since 2013. © Jörg Abendroth / MPI for Developmental Biology

Apoptosis is a natural, controlled suicide programme in multicellular organisms that eliminates cells which would otherwise be harmful to the body, such as extremely damaged or old cells. Biochemical reaction cascades lead to cell changes and death. Cellular waste can be removed by macrophages or other immune system cells. A key step in apoptosis is the mitochondrial release of the pro-apoptotic factor cytochrome C into the cytoplasm where cytochrome C can interact with factors different from the ones found in the mitochondria. If this interaction occurs, programmed cell death cannot be stopped. Manipulating the release of cytochrome C into the cytoplasm would, therefore, be a perfect medical target. On the one hand, targeting this particular process would prevent the passage of cytochrome C in cases where apoptosis is erroneously induced due to disease. On the other hand, in diseases such as cancer where apoptosis is suppressed, enabling abnormal cells to multiply and damage the entire organism, a drug to drive abnormal cells into apoptosis would be welcome.

A team of scientists from Tübingen led by Prof. Dr. Ana J. García-Sáez has managed to elucidate the mechanism of cytochrome C release and identify targets for therapeutic interventions. García-Sáez carries out research work at the IFIB (Interfaculty Institute of Biochemistry) at the University of Tübingen as well as at the Max Planck Institute for Intelligent Systems in Tübingen. She has been working on the biophysics of membranes and protein-membrane interactions for many years. Her latest results were obtained thanks to a European Research Council (ERC) Starting Grant of around 1.4 million euros, which she was awarded in 2012. The funding period will end in 2017.

The formation of membrane pores is crucial for apoptosis

García-Sáez works with artificial membrane systems in which a single lipid bilayer rests on a glass support. Bax proteins migrate into the membrane where they aggregate into pores that are large enough for the pro-apoptotic factor cytochrome C to pass through. © Katia Cosentino, IFIB University of Tübingen

The release of cytochrome C into the cytoplasm relies on pore formation in the outer mitochondrial membrane. Bcl-2 proteins are key in this process, in particular, a protein called Bax. García-Sáez and her team used artificial membrane systems to show that Bax proteins are integrated into the membrane as monomers, thereby forming pores. "We produced human Bax proteins in bacteria and added them to synthetic mitochondrial membranes," says García-Sáez "The proteins mostly assemble into dimers in the membranes, but we have also found tetramers and hexamers. We also assume that octamers and decamers are produced, but they are difficult to detect. The size of the pores in our artificial membrane system is highly variable and changes with the amount of Bax proteins added. The more Bax proteins present in the membrane, the larger the pores," says García-Sáez.

Mitochondria are surrounded by two membranes; Bax proteins are found in the outer membrane. The pores are not very selective and the theoretical possibility exists that other molecules could also pass through the membranes. Equally feasible is that proteins could be transported in the opposite direction. It is still unclear what this implies for apoptosis. García-Sáez explains: "Mitochondria undergo major changes during apoptosis, but these processes are not yet fully understood." The exact role of the membrane in the oligomerisation of the Bax proteins and hence the initiation of pore formation also needs to be explored. It is, however, clear that pore formation is controlled by proteins belonging to the Bcl-2 family. And this is where medical applications could be possible.

García-Sáez has managed to identify proteins that promote the aggregation of Bax proteins and others that prevent the formation of Bax oligomers. "In healthy cells, inactive Bax monomers are found in the cytoplasm. We have been able to show that cBid, which is another protein from the Bcl-2 family, has no influence on the oligomerisation process. The protein Bcl-xL can inhibit Bax by breaking Bax oligomers into di- and monomers," says García-Sáez. Even though many details are not yet understood, the researchers' work will contribute to the development of molecules that inhibit or promote the oligomerisation of Bax proteins and hence pore formation, which would then interfere with apoptotic processes. Other researchers have shown that small peptides derived from Bax or Bcl-2 domains are able to induce the permeabilisation of membranes and hence apoptosis.

Drugs can be derived from regulatory proteins

Schematic drawing of what happens at the outer mitochondrial membrane: cBid and Bax belong to the same protein family. cBid can activate Bax monomers at the outer mitochondrial membrane. The monomers are then integrated into the membrane where they form dimers. Dimers can aggregate into larger Bax oligomers and generate different sized pores. © Katia Cosentino, IFIB University of Tübingen

This knowledge is potentially groundbreaking for the development of therapeutic substances. "However, the metabolic processes involved in pore formation and the induction of apoptosis are nevertheless far more complicated than initially expected. Although some small molecules are already being tested in clinical trials, it is difficult to develop molecules that target specifically diseased cells," says García-Sáez. García-Sáez nevertheless believes one day apoptotic processes will be controlled with small molecules that target pore formation. "Theoretically, such molecules have the potential to treat all diseases that result from defective apoptotic processes, including cancer, neurodegenerative diseases and cardiac infarction," García-Sáez who, together with her team, will continue to elucidate the fundamental mechanisms and control processes of pore formation in order to create the necessary basis for developing such proteins.

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