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Immunosuppressants / Apoptosis / Bioenergy / Memory formation

Dr A. Lawen

Apoptosis

The aim of our research on apoptosis is to better understand the mechanisms involved in the induction of apoptosis, a non-inflammatory form of programmed cell death, which plays an important role in development of both the nervous and the immune system, in cancer and in autoimmune diseases. We have shown that bioenergy perturbance by both inhibitors of mitochondrial function and the PMOR system (see below) readily induces apoptosis. Use of immunosuppressants revealed the role that a specific protein phosphatase (calcineurin) plays in the signal transduction (Cell Growth Differ. 7 (1996) 1315-1325). A specific aim of this project is to further the understanding of the action of the 'anti-apoptotic' proto-oncogene product Bcl-2. We are also studying the effect of didemnin B on cancerous blood cells. This drug has been developed by Upjohn Ltd. It is at present in the clinical phase II trial in the USA as a drug for solid tumor treatment. We have shown that it is a very strong and rapid inducer of apoptosis, preferentially affecting cancer cells. We are now investigating its potential as a possible anti-leukemia drug. We have shown that didemnin B causes a very rapid onset of apoptosis in some cell lines (Biochem. Biophys. Res. Commun. 215 (1995) 1130-1136) and that its signalling involves tyrosine phosphorylation events (FEBS Lett. 383 (1996) 1-5). We are trying to establish the exact signalling pathways involved in all three systems of apoptosis and to identify the cellular didemnin B receptor. We have shown that didemnin B induces apoptosis through the "intrinsic" pathway (Oncogene 20, 4085-4094).

The general death executioners are cysteine aspartic acid proteases (caspases).  Two general pathways of caspase-activation exist: a mitochondrial pathway and a receptor pathway.  The mitochondrial pathway leads to release of cytochrome c into the cytosol, where it binds to Apaf-1.  Procaspase-9 (an initiator caspase) is then recruited to form the apoptosome.  The apoptosome-bound procaspase-9 is activated and can then activate an effector caspase (caspase-3) which can do the final job.  The relative levels of pro- and antiapoptotic members of the Bcl-2 family of proteins determine over susceptibility for apoptosis.

There exist two principal views on the importance of cytochrome c release and the mitochondria; in one hypothesis, it is the major control of the intrinsic pathway, in the other it merely functions as amplification loop for caspase activation.

Our data (Oncogene 20, 4085-4094) would suggest the latter view to be the more accurate one and we intend to further our investigations into this aspect.

Plasma membrane NADH-oxidoreductase (PMOR)

We have also commenced to further characterise the PMOR system (J. Bioenerg. Biomembr. 28 (1996) 531-540). We were able to show that its up-regulation is a prerequisite for the survival of mitochondrially deprived human cells (J. Biol. Chem. 269 (1994) 30097-30100). We have identified the plasma membrane NADH:ferricyanide reductase and identified and characterised it on the molecular level. Cells over-expressing the enzyme show reduced levels of apoptosis. We are trying to establish the exact role this enzyme system plays in regulating the cellular energy hosehold (which may contribute to the understanding of mitochondrial diseases and possibly aging) and to further characterise the enzyme system. 

Biosynthesis of the immunosuppressant cyclosporin A (CyA)

CyA is a cyclic undecapeptide and is widely used as immunosuppressant in human transplantation surgery to prevent graft rejections, the major post-operative problem in transplantation medicine. We discovered, purified and characterised the enzyme responsible for its biosynthesis (reviewed in Prog. Med. Chem. 33 (1996) 53-97). The enzyme called 'cyclosporin synthetase' (J. Biol. Chem. 265 (1990) 11355-11360), is the most complex enzyme known: it is a single polypeptide chain with a molecular mass of about 1.7 MDa and can be viewed as a huge 'superenzyme', comprised of at least 40 'normal' enzymes. We are using this enzyme for studying the possibilities of constructing new enzymes with new activities. Our present goals are to establish (1) the molecular shape of cyclosporin synthetase and (2) its subcellular localisation in the fungus by EM studies and (3) the crystal structure of the enzyme. This will further the understanding of the mechanism of biosynthesis of CyA (J. Biol. Chem. 269 (1994) 2841-2846). We also plan (4) to isolate by partial proteolysis and to characterise single domains of the enzyme and possibly of related enzymes in order to compare their activities with those in the intact enzyme. Finally, we plan (5) to unravel the folding sequence of the domains of cyclosporin synthetase and to establish with this enzyme and its domains a model system for reversible denaturation of multidomain enzymes (FEBS Lett. 380 (1996) 157-160).

Biochemistry of memory formation (Collaboration with Prof KT Ng Dept Psychol.)

The immunophilins (cyclophilins and FKBP's) are binding proteins of the immunosuppressants (CyA and FK-506). As complexes with these compounds they are inhibitors of the calcium-dependent protein (Ser) phosphatase calcineurin. As uncomplexed proteins they exert an enzymatic activity known as 'peptidyl-prolyl-cis/trans-isomerase (PPIase)'. The activity is inhibited by the immunosuppressants. It has been shown that calcineurin (Brain Res. 730 (1996) 107-117) and cyclophilin function (FEBS Lett.,431, (1998) 386-390, Brain Res. 927, 180-194.) are needed for long-term memory function in chicks. We are interested to establish the role of both protein kinases/phosphatases and PPIases in memory formation.

Project Areas

  1. Apoptosis, a distinct form of cell death: its induction by energy pertubation and cytotoxic drugs; its regulation and signalling pathways involved (Dr A. Lawen) 
  2. The plasma membrane NADH-oxidoreductase system: characterisation and cellular actions (Dr A. Lawen) 
  3. Biosynthesis and mechanism of action of immunosupressants (Dr A. Lawen) 
  4. The role of protein kinases/phosphates and peptidyl-prolyl-cis/trans-isomerases in long - term memory formation of chicken (Dr A. Lawen and Professor K.T. Ng (Dept Psychology))