Proteome-wide quantitative analysis of redox cysteine availability in the Drosophila melanogaster eye reveals oxidation of phototransduction machinery during blue light exposure and age.

The retina is one of the highest oxygen-consuming tissues because visual transduction and light signaling processes require large amounts of ATP. Thus, because of the high energy demand, oxygen-rich environment, and tissue transparency, the eye is susceptible to excess production of reactive oxygen species (ROS) resulting in oxidative stress. Oxidative stress in the eye is associated with the development and progression of ocular diseases including cataracts, glaucoma, age-related macular degeneration, and diabetic retinopathy. ROS can modify and damage cellular proteins, but can also be involved in redox signaling. In particular, the thiol groups of cysteines can undergo reversible or irreversible oxidative post-translational modifications (PTMs). Identifying the redox-sensitive cysteines on a proteome-wide scale provides insight into those proteins that act as redox sensors or become irreversibly damaged upon exposure to oxidative stress. In this study, we profiled the redox proteome of the Drosophila eye under prolonged, high intensity blue light exposure and age using iodoacetamide isobaric label sixplex reagents (iodo-TMT) to identify changes in cysteine availability. Although redox metabolite analysis of the major antioxidant, glutathione, revealed similar ratios of its oxidized and reduced form in aged or light-stressed eyes, we observed different changes in the redox proteome under these conditions. Both conditions resulted in significant oxidation of proteins involved in phototransduction and photoreceptor maintenance but affected distinct targets and cysteine residues. Moreover, redox changes induced by blue light exposure were accompanied by a large reduction in light sensitivity that did not arise from a reduction in the photopigment level, suggesting that the redox-sensitive cysteines we identified in the phototransduction machinery might contribute to light adaptation. Our data provide a comprehensive description of the redox proteome of Drosophila eye tissue under light stress and aging and suggest how redox signaling might contribute to light adaptation in response to acute light stress.

The interaction between the Dbf4 ortholog Chiffon and Gcn5 is conserved in Dipteran insect species.

Chiffon is the sole Drosophila ortholog of Dbf4, the regulatory subunit for the cell-cycle kinase Cdc7 that initiates DNA replication. In Drosophila, the chiffon gene encodes two polypeptides with independent activities. Chiffon-A contains the conserved Dbf4 motifs and interacts with Cdc7 to form the Dbf4-dependent Kinase (DDK) complex, which is essential for a specialized form of DNA replication. In contrast, Chiffon-B binds the histone acetyltransferase Gcn5 to form the Chiffon histone acetyltransferase (CHAT) complex, which is necessary for histone H3 acetylation and viability. Previous studies have shown that the Chiffon-B region is only present within insects. However, it was unclear how widely the interaction between Chiffon-B and Gcn5 was conserved among insect species. To examine this, we performed yeast two-hybrid assays using Chiffon-B and Gcn5 from a variety of insect species and found that Chiffon-B and Gcn5 interact in Diptera species such as Australian sheep blowfly and yellow fever mosquito. Protein domain analysis identified that Chiffon-B has features of acidic transcriptional activators such as Gal4 or VP16. We propose that the CHAT complex plays a critical role in a biological process that is unique to Dipterans and could therefore be a potential target for pest control strategies.

Chiffon triggers global histone H3 acetylation and expression of developmental genes in Drosophila embryos.

The histone acetyltransferase Gcn5 is critical for gene expression and development. In Drosophila, Gcn5 is part of four complexes (SAGA, ATAC, CHAT and ADA) that are essential for fly viability and have key roles in regulating gene expression. Here, we show that although the SAGA, ADA and CHAT complexes play redundant roles in embryonic gene expression, the insect-specific CHAT complex uniquely regulates expression of a subset of developmental genes. We also identify a substantial decrease in histone acetylation in chiffon mutant embryos that exceeds that observed in Ada2b, suggesting broader roles for Chiffon in regulating histone acetylation outside of the Gcn5 complexes. The chiffon gene encodes two independent polypeptides that nucleate formation of either the CHAT or Dbf4-dependent kinase (DDK) complexes. DDK includes the cell cycle kinase Cdc7, which is necessary for maternally driven DNA replication in the embryo. We identify a temporal switch between the expression of these chiffon gene products during a short window during the early nuclear cycles in embryos that correlates with the onset of zygotic genome activation, suggesting a potential role for CHAT in this process. This article has an associated First Person interview with the first author of the paper.