Dr Alfons Lawen

Senior Lecturer

Telephone: +61 3 9905 3711 Facsimile: +61 3 9905 4699 Email: Alfons.Lawen@med.monash.edu.au Group members 2009: Dr Darius Lane Mr Po-Hua Chang Mr Samuel Thompson

German version

Career:

I did a diploma in chemistry and a PhD (Dr. rer. nat.) in biochemistry, both at the University of Würzburg, West-Germany. After postdoctoral studies at the Technical University in Berlin I have there established my own research group. I was appointed a Lecturer at the Department of Biochemistry and Molecular Biology in March 1993, where my current position is that of a Senior Lecturer.

Research:

I have published extensively on the biosynthesis of the immunosuppressant cyclosporin A. After joining Monash Biochemistry Department in March 1993, I also started to work on an enzyme system located in the plasma membrane of human cells, on apoptosis and - in collaboration with Prof. Kim Ng and his group (from the Department of Psychology) on the Biochemistry of memory formation.

Our major research area concerns the plasma membrane NADH-oxidoreductase (PMOR),electron transport across the plasma membrane and its involvement in iron import into mammalian cells.

Iron is important for cellular survival and without it, every cell will die. Proteins involved in iron uptake are up-regulated in cancer cells and inhibiting iron uptake can inhibit their growth. In its physiological form, extracellular iron is complexed by chelators, molecules that bind to metallic ions. The most important iron chelators are the protein transferrin and the metabolite citrate. In order for iron from iron citrate to be taken up by a cell, it has to be first reduced from iron (III) to iron (II). For this reduction to occur, the electrons must be supplied from inside the cell. We have analysed the cellular uptake of iron from iron citrate (Fe3+-Cit) in detail, and found that vitamin C is needed to reduce iron and to take it up. Cells actively export reduced vitamin C (ascorbate, Asc) for this purpose and take up oxidised vitamin C (dehydroscorbic acid, DHA). The cells then reduce it back to ascorbate and release it for further iron reduction (see figure). In this way, vitamin C from the cells supplies electrons for this reaction and the subsequent uptake of iron.

Ascorbate/DHA shuttling in mammalian NTBI uptake. Recent evidence suggests that NTBI ferrireduction may occur by transplasma membrane Asc cycling in which i) extracellular Asc reacts directly with NTBI, forming both DHA and Fe2+. The latter is then imported into the cell putatively via ferrous-selective transporters (e.g. DMT1 and/or Zip14). Extracellular Asc is subsequently regenerated for further ferric reduction events by ii) DHA import via glucose  transporters (GLUTs), iii) intracellular reduction of DHA to Asc by an unspecified redox couple (‘R/O’; e.g. GSH/GSSG or NADPH/NADP+), followed by release of Asc through as yet unidentified Asc transporters (Anion Channel) in the plasma membrane (PM).

Recent Publications:

1. Lane, D.J.R. and Lawen, A. (2009): Ascorbate and plasma membrane electron transport – enzymes vs. efflux.  Free Rad. Biol. Med. 47, 485-495
2. Lane, D.J.R. and Lawen, A. (2009): Transplasma membrane electron transport comes in two flavors. Biofactors 34, 191-200.
3. Lane, D.J.R. and Lawen, A. (2008): Non-transferrin iron reduction and uptake are regulated by transmembrane ascorbate cycling in K562 cells. J.Biol. Chem. 283, 12701-12708.
4. Lane, D.J.R. and Lawen, A. (2008): A highly sensitive colorimetric microplate ferrocyanide assay applied to ascorbate-stimulated transplasma membrane ferricyanide reduction and mitochondrial succinate oxidation. Anal. Biochem. 373, 287-295.
5. Lawen, A., Ly, J. D. Lane, D.J.R., Zarschler, K. Messina, A. and De Pinto, V. (2005): Voltage-dependent anion-selective channel 1 (VDAC1) - a mitochondrial protein, rediscovered as a novel enzyme in the plasma membrane. Invited review, Int. J. Biochem. Cell Biol. - Molecules in Focus 37, 277-282.
6. Baker, M. A., Lane, D.J.R., Ly, J. D., De Pinto, V. and Lawen, A. (2004): Voltage dependent anion channel 1 is an NADH:ferricyanide reductase. J. Biol. Chem. 279, 4811-4819.
7. De Pinto, V., Messina, A., Accardi, R., Aiello, R., Guarino, F., Tommasello, M., Tommasino, M., Tasca, G., Casadio, R., Benz, R., De Giorgi, F., Ichas, F., Baker, M. and Lawen, A. (2003): New functions of an old protein: the eukaryotic porin or voltage dependent anion selective channel (VDAC). Ital. J. Biochem. 52, 17-24.
8. Malik, S. G., Vaillant, F. and Lawen, A. (2004): Plasma membrane NADH-oxidoreductase in cells carrying mitochondrial DNA G11778A mutation and in cells devoid of mitochondrial DNA (rho0). BioFactors 20, 189-198.
9. Baker, M. A., Ly, J. D. and Lawen, A. (2004): Characterization of VDAC1 as a plasma membrane NADH-oxidoreductase.  BioFactors 21,215-221.
10. Macho, A., Sancho, R., Minassi, A., Appendino, G., Lawen, A. and Muñoz, E. (2003): Involvement of reactive oxygen species in capsaicinoid-induced apoptosis in transformed cells. Free Radical Res. 37, 611-619. 
11. Ly, J. D. and Lawen, A. (2003): Transplasma membrane electron transport: enzymes involved and biological function. Redox Report 8, 3-21.
12. Baker, M. A. and Lawen, A. (2000): The function of the plasma membrane NADH-oxidoreductase system. A critical review of the structural and functional data. Antioxidants and Redox Signaling, Antioxid. Redox Signal. 2, 197-212. 
13. Lawen, A., Baker, M. A. and Malik, S. (1998): Apoptosis and redox homeostasis - On a possible mechanism of action of Bcl-2. Protoplasma 205, 10-20.
14. Vaillant, F., Larm, J. A., McMullen, G.L., Wolvetang, E.J. and Lawen, A. (1996): Effectors of the mammalian plasma membrane NADH-oxidoreductase system. Ubiquinone analogues as potent stimulators. J. Bioenerg. Biomembr. 28, 531-540.
15. Wolvetang, E. J., Larm, J. A., Moutsoulas, P. and Lawen, A. (1996): Apoptosis induced by inhibitors of the plasma membrane NADH-oxidase involves Bcl-2 and calcineurin. Cell Growth & Differ. 7, 1315-1325.
16. Larm, J. A., Vaillant, F., Linnane, A. W. and Lawen, A. (1994): Up-regulation of the plasma membrane oxidoreductase as a prerequisite for the viability of human Namalwa rho^o cells. J. Biol. Chem. 269, 30097-30100.

 Previous research, not presently pursued

We were using the system of induction of apoptosis by didemnin B in HL-60 cells for unravelling some minimal requirements for apoptosis to occur. Specifically we were investigating the role played in this system by 1) the mitochondrial membrane potential, 2) the mitochondrial transition, 3) caspases and 4) the Bcl-2 protein family.

Recent Publications:

1. Berridge, M. V., Herst, P. M. and Lawen, A. (2009): Targeting mitochondrial permeability in cancer drug development. Mol. Nutr. Food Res. 53, 76-86.
2. Lawen, A. (2007): Another piece of the puzzle of apoptotic cytochrome c release. Mol. Microbiol. 66, 553-556.
3. Ralph, S. J., Low, P., Dong, L, Lawen, A. and Neuzil, J. (2006): Mitocans: mitochondria targeted anti-cancer drugs as improved therapies and related patents. Recent Patents Anti-Canc. Drug Disc. 1, 327-346.
4. Lawen, A. (2003): Apoptosis - an introduction. BioEssays 25, 888-896.
5. Ly, J. D., Grubb, D.R. and Lawen, A. (2003): The mitochondrial membrane potential (Delta Psim) in apoptosis; an update. Apoptosis 8, 115-128.
6. Baker, M. A., Grubb, D. R. and Lawen, A. (2002): Didemnin B induces apoptosis in proliferating but not resting peripheral blood mononuclear cells.Apoptosis 7, 407-412.
7. Grubb, D. R., Ly, J. D., Vaillant, F., Johnson, K.L. and Lawen, A. (2001): Mitochondrial cytochrome c release is caspase-dependent and does not involve mitochondrial permeability transition in didemnin B-induced apoptosis. Oncogene 20, 4085-4094.
8. Johnson, K.L. and Lawen, A. (1999): Rapamycin inhibits didemnin B-induced apoptosis in human HL-60 cells: evidence for a possible involvement of FKBP-25. Immunol. Cell Biol. 77, 242-248.
9. Johnson, K.L., Grubb, D. R. and Lawen, A. (1999): Unspecific activation of caspases during the induction of apoptosis by didemnin B in human cell lines. J. Cell. Biochem. 72, 269-278.
10. Johnson, K. L., Vaillant, F. and Lawen, A. (1996): Protein tyrosine kinase inhibitors prevent didemnin B-induced apoptosis in HL-60 cells. FEBS Lett. 383, 1-5.
11. Grubb, D., Wolvetang, E.J. and Lawen, A. (1995): Didemnin B induces cell death by apoptosis. The fastest induction of apoptosis ever described. Biochem. Biophys. Res. Commun. 215, 1130- 1136.

During my time in Berlin I worked intensively on the biosynthesis of the important immunosuppressive drug cyclosporin A. I showed that this compound is synthesised by the largest multienzyme known (cyclosporin synthetase), which does not consist of subunits (1.7 MDa). One aim of my work was the use of the enzyme for the synthesis of new cyclosporin A analogues which are more suitable for other uses than immunosuppression. This work was done in close cooperation with Sandoz Pharma Ltd, Basel, Switzerland.

Recent Publications:

1. Velkov, T. and Lawen, A. (2003): Mapping and molecular modeling of S-adenosyl-L-methionine binding site in N-methyltransferase domains of the multifunctional polypeptide cyclosporin synthetase. J. Biol. Chem. 278, 1137-1148.
2. Velkov, T. and Lawen, A. (2003): Nonribosomal peptide synthetases as technological platforms for the synthesis of highly modified peptide bioeffectors - cyclosporin synthetase as a complex example. Biotechnol. Annu. Rev. 9, 151-197.
3. Velkov, T. and Lawen, A. (2003): Photoaffinity labeling of the N-methyltransferase domains of cyclosporin synthetase. Photochem. Photobiol. 77, 129-137.
4. Lawen, A. (1996): Biosynthesis and mechanism of action of cyclosporins. Prog. Med. Chem. 33 chapter 2, 53-97.
5. Dittmann, J., Vaillant, F., Kleinkauf, H. and Lawen, A. (1996): Reversible denaturation of cyclosporin synthetase by urea. FEBS Lett. 380, 157-160.
6. Traber, R., Kobel, H., Loosli, H.-R., Senn, H., Rosenwirth, B. and Lawen, A. (1994): [MeIle4]Cyclosporin, a novel natural cyclosporin with anti-HIV activity: Structure elucidation and biosynthesis. Antivir. Chem. Chemother. 5, 331-339.
7. Dittmann, J., Wenger, R. M., Kleinkauf, H. and Lawen, A. (1994): Mechanism of biosynthesis of cyclosporin A: Evidence for synthesis via a single linear undecapeptide precursor.J. Biol. Chem. 269, 2841-2846.

The aim in our project into the biochemistry of memory formation (in collaboration with the group of Prof. Kim T. Ng, Dept Psychol.) was to examine in more detail the role of Ca²⁺ -regulated protein phosphorylation/dephosphorylation and peptidyl-prolyl-cis/trans-isomerases in memory formation, arguably the most important cognitive function carried out by the brain. Our preliminary results strongly imply that cyclophilin activity is required for the formation of a relatively permanent stage of memory in the day-old chick and constitute one of the few sets of data establishing a physiological role for a cyclophilin.

Recent Publications:

1. Bennett, P. C., Moutsoulas, P., Lawen, A., Perini, Eloise and Ng, K. T. (2003): Novel effects on memory observed following unilateral intracranial administration of okadaic acid, cyclosporin A, FK506 and [MeVal4]CyA. Brain Res. 988, 56-68.
2. Bennett, P. C., Schmidt, L., W., Lawen, A., Moutsoulas, P. and Ng, K. T. (2002): Cyclosporin A, FK506 and rapamycin produce multiple, temporally distinct, effects on memory following passive avoidance training in the chick. Brain Res. 927, 180-194.
3. Singaretnam, L.G., Bennett, P.C., Zhao, W-Q., Ng, K.T. and Lawen, A. (2000): Peptidyl-prolyl-cis/trans - isomerase activity may be neccessary for memory formation. In: Immunophilius in the brain. FKBP-Ligands: Novel Strategies for the Treatment of Neurodegenerative Disorders. Prous Science ISBN 84-8124-165-2; pp 75-89.
4. Zhao, W., Lawen, A. and Ng, K.T. (1999): Changes in phosphorylation of Ca²⁺/calmodulin -dependent protein kinase II (CaMKII) in processing of short-term and long-term memories following passive avoidance learning. J. Neurosci. Res. 55, 557-568.
5. Bennett, P. C., Singaretnam, L. G., Zhao, W.-Q., Lawen, A. and Ng, K. T. (1998): Peptidyl-prolyl-cis/trans-isomerase activity may be necessary for memory formation. FEBS Lett. 431, 386-390.
6. Bennett, P. C., Zhao, W.-Q., Lawen, A. and Ng, K. T. (1996): Cyclosporin A, an inhibitor of calcineurin, impairs memory formation. Brain Res. 730, 107-117.