In Vivo Effects of Methionine Sulfoxide Reductase Deficiency in Drosophila melanogaster

The deleterious alteration of protein structure and function due to the oxidation of methionine residues has been studied extensively in age-associated neurodegenerative disorders such as Alzheimer’s and Parkinson’s Disease. Methionine sulfoxide reductases (MSR) have three well-characterized biological functions. The most commonly studied function is the reduction of oxidized methionine residues back into functional methionine thus, often restoring biological function to proteins. Previous studies have successfully overexpressed and silenced MSR activity in numerous model organisms correlating its activity to longevity and oxidative stress. In the present study, we have characterized in vivo effects of MSR deficiency in Drosophila. Interestingly, we found no significant phenotype in animals lacking either methionine sulfoxide reductase A (MSRA) or methionine sulfoxide reductase B (MSRB). However, Drosophila lacking any known MSR activity exhibited a prolonged larval third instar development and a shortened lifespan. These data suggest an essential role of MSR in key biological processes.

(Supplemental Figure 2). The lifespan assay was repeated in the presence or absence of 2mM paraquat (as used in Figure 5 in the manuscript) with these four lines. There were no significant differences in the four lifespan curves (data not shown).

Supplemental Figure 2: Longevity of MSRA Deletion and Wildtype Revertants.
Lines 16671 w16 , 16671 w45 , 16671 w72 are MSRA wildtype due to precise excision of the P-element, whereas 16671 w90 is a null mutant due to ~1.4 Kbp deletion of the MSRA locus. The results are the average number of flies living after transfer to normal culture medium. A total of 50 flies (five vials with 10 flies each) were used for each strain. The strains used were 16671 w16 (wildtype control ), 16671 w45 (wildtype control ), 16671 w72 (wildtype control ) and 16671 w90 (MSRA deletion ). The error bars are standard error of the mean.
After determining there were no phenotypic differences between the MSRA deletion lines and the controls, the MSRA null line 16671 w90 was used as one of the parental strains for creating the double MSR deletion line (described below).

Creation of the MSRB deletion line
Stock 17116 having a P-element in the MSRB locus was obtained from the Bloomington Stock Center (Bloomington, IN). The P-element was inserted in the proximal promoter region of the MSRB gene, just a few bases upstream of the transcriptional start site.
The stock was received as a heterozygous stock, having the TM6B balancer for chromosome 3 because it was homozygous lethal. Recombination with a wild-type stock designated Biocore, a white eyed wildtype line from the University of Wisconsin-Madison [1], removed the recessive lethal mutation, and the stock became fully viable when homozygous for the P-element insertion. We did not use the YW stock for this isogenization process because our YW stock line had become contaminated and we had the Biocore wildtype line available from our colleague Ken Dawson-Scully. The stock originated from the Bloomington Stock Center (Bloomington, IN; Flybase ID FBal0033148). After isogenization with Biocore, the 17116 P-element line exhibited a small, but detectable amount of MSRB mRNA using RT-PCR. Anecdotally, the line still seemed not as "robust" as the YW wildtype lines. We felt this might be due, at least in part, to the dramatically shorter lifespan of the Biocore line compared to the YW wildtype strain, which we obtained a second time from the Rodney Murphey lab (FAU, Boca Raton, FL) since our original stock, as noted previously, had become contaminated (Supplemental Figure 3). The 17116 stock was backcrossed to the newly acquired YW line for several generations.

Supplemental Figure 3: Lifespan differences between the YW and Biocore wildtype lines.
The wildtype line designated Biocore had a statistically significant decreased lifespan compared with the wildtype line "YW" (p < 0.001, log-rank test for survival). The maximum lifespan of the YW line was 85 days compared with 46 for the Biocore line. The median lifespan was 64 versus 39 days for each line respectively.
Using the same methodology employed in the creation of the MSRA deletion lines, the YW isogenized 17116 line was exposed to transposase to create an MSRB deletion line. We recovered three lines containing a ~2.5 kb deletion in MSRB extending from a site just upstream of the transcriptional start site into the third exon, which is shared by all isoforms (Supplemental Figure 4). Homozygous lines for each of these three MSRB deletions showed no detectable transcript or protein using RT-PCR and Western blotting, respectively (data not shown).
The three MSRB deletion lines (17116 w2 , 17116 w26 , and 17716 w92 ) were compared to the recovered YW wildtype line in a lifespan assay. The survival curves were similar to one another with the median survival for all the lines between 37.5 and 43 days, but line 17716 w92 had a survival curve that was statistically indistinguishable from the YW wildtype line and was selected to use in creating the MSR double deletion line described below (Supplemental Figure 5). The MSRB deletion lines designated 17116 w2 and 17116 w26 were statistically different than the YW control line (p = 0.003 and p > 0.0001, respectively, log-rank test for survival), but line 17116 w92 was statistically identical to the YW control (p = 0.6186, log-rank test of survival).