Abstract
We cloned cDNA for the Bombyx mori DJ-1 protein (BmDJ-1) from the brains of larvae. BmDJ-1 is composed of 190 amino acids and encoded by 672 nucleotides. Northern blot analysis showed that BmDJ-1 is transcribed as a 756-bp mRNA and has one isoform. Reverse transcriptase (RT)-PCR experiments revealed that the BmDJ-1 was present in the brain, fatbody, Malpighian tubule, ovary and testis but present in only low amounts in the silkgland and hemocyte of day 4 fifth instar larvae. Immunological analysis demonstrated the presence of BmDJ-1 in the brain, midgut, fatbody, Malpighian tubule, testis and ovary from the larvae to the adult. We found that BmDJ-1 has a unique expression pattern through the fifth instar larval to adult developmental stage. We assessed the anti-oxidative function of BmDJ-1 using rotenone (ROT) in day 3 fifth instar larvae. Administration of ROT to day 3 fifth instar larvae, together with exogenous (BmNPV-BmDJ-1 infection for 4 days in advance) BmDJ-1, produced significantly lower 24-h mortality in BmDJ-1 groups than in the control. 2D-PAGE revealed an isoelectric point (pI) shift to an acidic form for BmDJ-1 in BmN4 cells upon ROT stimulus. Among the factors examined for their effects on expression level of BmDJ-1 in the hemolymph, nitric oxide (NO) concentration was identified based on dramatic developmental stage-dependent changes. Administration of isosorbide dinitrate (ISDN), which is an NO donor, to BmN4 cells produced increased expression of BmDJ-1 compared to the control. These results suggest that BmDJ-1 might control oxidative stress in the cell due to NO and serves as a development modulation factor in B. mori.
Highlights
The protein DJ-1 is ubiquitously expressed in cells and it is highly conserved across a wide variety of organisms, showing moderate sequence identity with heat shock protein 31 (HSP31) chaperones and ThiJ/PfpI cysteine proteases [1]
Characterization of the B. mori variant, Bombyx mori DJ-1 protein (BmDJ-1), by cDNA cloning from the brains of the fifth instar larvae shows the presence of Cys and Leu, which are key residues for the function of DJ-1
On a phylogenetic tree of DJ-1 proteins, two orthologs of D. melanogaster, DJ-1a and DJ-1b, and BmDJ-1 placed in distinct clusters
Summary
The protein DJ-1 is ubiquitously expressed in cells and it is highly conserved across a wide variety of organisms, showing moderate sequence identity with heat shock protein 31 (HSP31) chaperones and ThiJ/PfpI cysteine proteases [1]. DJ-1, which is encoded by PARK7, is a multi-functional protein that plays roles in chaperoning, RNA-binding, SUMOylation, apoptosis, and protease activity [2]. Oxidative modification leads to mitochondrial damage in cultured cells exposed to 1methyl-4-phenyl-1,2,3,6 tetrahydropyridine (MPTP), 6-hydroxydopamine (6-OHDA), paraquat (PQ), and rotenone (ROT), which inhibit the mitochondrial electron transfer chain of mitochondrial complex I [4]. These compounds enhance production of reactive oxygen species (ROS) and reduce production of ATP, resulting in mitochondria dysfunction [5]. MPTP, 6-OHDA, PQ, and ROT are used to produce PD models in rats and Drosophila and to analyze the pathology of PD [6,7]
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