UNIVERSITY OF MALTA
LIFE SCIENCE RESEARCH SEMINARS
Web: http://www.um.edu.mt/events/scisem/
Email: scisem@um.edu.mt
Abstract form
Title: C. elegans MnSOD gateway mutants - from gene to protein structure
Presenter: Rosalin Bonetta
Contact address: Department of Physiology and Biochemistry, University of Malta,
Msida MSD2080.
Tel:
Fax:
Email: rosalin.bonetta@um.edu.mt
Presentation date: 13th May 2013
Abstract
Manganese superoxide dismutase (MnSOD) is an antioxidant enzyme that catalyses
the dismutation of superoxide radicals (O2-) to O2 and H2O2, providing protection in the
mitochondria against the oxidative stress generated during cellular respiration. In many
types of cancer, MnSOD demonstrates tumour suppressor properties. Studies by Davis et
al. (2004) on an active site H30N mutant of the human MnSOD have shown that this mutant
has anti-proliferative effects in vitro as well as anti-tumour effects in vivo, when overexpressed in the A549 lung carcinome.
Giglio et al. (1994) isolated, cloned and sequenced two MnSOD genes (sod-2 and
sod-3) from the model organism C. elegans. These MnSODs have been identified as
components of the longevity pathway that increase life-span tenfold. Preliminary kinetic
studies of these MnSODs have shown similarity to the anti-proliferative human H30N
mutant. One aim of this research project is to provide further insight into the physiological
function of the MnSOD-2 and MnSOD-3 isoforms of C. elegans MnSOD in terms of
antioxidant protection and regulation of cell signaling. A second aim is to investigate the
structural and catalytic role of the gateway residue at the H30 position through site directed
mutagenesis especially in the context of anti-proliferative properties.
Three H30 active site mutants of MnSOD-3 were generated, sequenced and overexpressed in Escherichia coli. The recombinant proteins were purified using a combination
of affinity chromatography and gel filtration chromatography. The activity of the pure
proteins was measured and the physiochemical properties of the wild type MnSODs and the
three mutants were characterised.
X-ray crystallography was performed at the Diamond Light Source, Harwell and
University of Leeds in collaboration with Dr. Trinh. Further studies involved high field
electron paramagnetic resonance (HFEPR) in collaboration with Prof Un at CEA – Saclay
(France) to determine the electron spins of the manganese ion at the active site of the
protein in order to gain an understanding of its atomic and sub-atomic environment.