Download Effects of MethionineSulfoxide Reductase (Msr)

Graduate Student Research Day 2012
Florida Atlantic University
CHARLES E. SCHMIDT COLLEGE OF SCIENCE
Effects of MethionineSulfoxide Reductase (Msr) Deficiency in Drosophila melanogaster
Lindsay Bruce
Department of Biological Sciences, Charles E. Schmidt College of Science, Florida Atlantic
University, Boca Raton, FL
The overarching goal of my research is to investigate the role of oxidative stress in aging
and age-associated neurodegenerative diseases. There is clear evidence that antioxidant enzymes
play a role in aging. Methionine sulfoxide reductases (Msr) are a family of antioxidant enzymes
that reduce free and bound oxidized methionine. Two forms of Msr - MsrA and MsrB - are
specific for the S and R enantiomers of methionine sulfoxide, respectively. There are three wellcharacterized functions of Msr: It repairs oxidized methionine, acts as a catalytic antioxidant,
and regulates certain protein functions.
Previous research from our lab showed that complete loss of MsrA activity due to a loss
of function (LOF) genetic mutation had no affect on lifespan, oxidative stress resistance, body
mass, climbing ability, thermal stress, or fertility in Drosophila (unpublished data). This result
was unexpected since similar mutations in other organisms had shown clear phenotypes. To
obtain a clearer understanding of the biological effects of both MsrA and MsrB in Drosophila,
we created Drosophila lines with LOF mutations in both the MsrA and MsrB genes. We found
that MsrALOF BLOF flies have shorter lifespan compared with MsrA+ B+ (wild-type) flies.
Concomitantly, there is an increase in the oxidative damage to proteins in the strains completely
deficient in Msr activity. Further research to confirm the shortened lifespan and increased
protein oxidation will be pursued using RNA interference (RNAi). The underlying biochemical
processes responsible for the shortened lifespan and protein oxidation will also be investigated.
Effects of Methionine Sulfoxide Reductase (Msr) Deficiency
in Drosophila melanogaster
Lindsay Bruce and David Binninger
Florida Atlantic University
Abstract
Methods
There is clear evidence that
antioxidant enzymes play a role in
aging. Methionine sulfoxide
reductases (Msr) are a family of
antioxidant enzymes that reduce free
and bound oxidized methionine. Two
forms of Msr - MsrA and MsrB - are
specific for the S and R enantiomers
of methionine sulfoxide, respectively.
There are three well-characterized
functions of Msr (recently reviewed in
Biochimica et Biophysica Acta)[1].
First, it repairs oxidized methionine by
reducing methionine sulfoxide (Met(o)) to methionine. Second, it can
serve as a catalytic antioxidant and,
thus, destroy reactive oxygen species
(ROS) before they are able to
damage key molecules within the cell.
Finally, Msr regulates certain protein
functions through the redox cycle.
Lifespan – Three independent lines
for each genotype (MsrA+B+ and
MsrALOFBLOF) were used. 40 males
per line (120 flies per genotype) were
sorted into vials of cornmeal media
with 10 flies each. New food was
provided twice weekly and
survivorship was assayed three times
weekly.
Methods
Fly Lines – Loss of function of both
MsrA and MsrB was obtained through
imprecise excision of p-elements
located in each gene. Recombination
of MsrALOF and MsrBLOF lines resulted
in MsrA+B+ and MsrALOFBLOF lines.
Molecular Analysis – The lines
created through recombination were
analyzed by genomic PCR and
western blotting.
Results
WT
ALOF
ALOF
Conclusion
ALOF
ALOFBLOF
MsrA
MsrB
Figure 3: Western blotting with MsrA
and MsrB primary antibodies
• Confirmation of lifespan with RNAi
lines directed at MsrA and MsrB
• Phenotypic assays, including anoxia
resistance and larval development
Results
• Figure 4: Lifespan of male MsrA+B+
and MsrALOFBLOF flies. MsrALOFBLOF
flies have a 18% shorter median
lifespan (p < 0.05).
Figure 1: ~1500bp deletion in MsrA
and ~2500bp deletion in MsrB
MsrA
MsrB
Lane 1 – 10,000 kb
Lane 2 – A? BLOF
Lane 3 – A+BLOF
Lane 4 – A+B+ (WT)
Lane 5 – A+B+ (WT)
Lane 6 – A+B+ (WT)
Lane 7 – ALOFB+
Lane 8 – A+BLOF
Lane 9 – ??
Lane 10 – A?BLOF
Figure 2: Genomic PCR analysis of
recombinant lines with MsrA and
MsrB primers.
• Loss of Msr leads to a significantly
shorter lifespan and increased protein
carbonyl levels
Future Direction
Protein Oxidation – Protein
carbonyls were assessed by
spectrophotometric quantitation at
360nm using 2,4-DNPH.
1 2 3 4 5 6 7 8 9 10
• Loss of Msr was achieved through
imprecise p-element excision and
confirmed through genomic PCR and
western blotting
• Biochemical assays, including ATP
determination and S/R enantiomer
accumulation
• Transgenic rescue of phenotypes
using the UAS-Gal4 system
References
Moskovitz, J., Methionine sulfoxide reductases: ubiquitous
enzymes involved in antioxidant defense, protein regulation,
and prevention of aging-associated diseases. Biochim
Biophys Acta, 2005. 1703(2): p. 213-219
Acknowledgements
Figure 5: MsrALOFBLOF flies show
increased protein carbonyls
compared with MsrA+B+ flies at 7
days post eclosion.
• NIH 2R15AG02256-02A1
• Drs. Herbert Weissbach, Rod Murphey, Ken Dawson-Scully,
Tanja Godenschwege, Kailiang Jia, and John Nambu for
helpful discussions.
• My lab mates, especially Karin Schey for Fig. 1