Integrative Computational and Experimental Analysis of Curly Su Mutations in Drosophila melanogaster

The Curly Su (dMPO) protein, a homolog of the human myeloperoxidase (hMPO), is critical for wing development in Drosophila melanogaster. Like human peroxidases, dMPO is involved in various cellular and physiological processes, producing significant quantities of reactive oxygen species that contribute to both development and immunity in the fruit fly. Given the significant sequence and structural similarities between dMPO and hMPO, dMPO serves an ideal model for studying peroxidase functions and related pathologies. We performed saturated computational mutagenesis on dMPO, analyzing the effects of 11,191 missense mutations on its stability. Notably, the G378W mutation exhibited the greatest destabilizing effect, while the W621R, potentially pathogenic, also reduced dMPO stability. To investigate these effects in vivo, we used genome editing to generate the transgenic Drosophila with G378W, W621R, and deletion of residues 305-687. Remarkably, G378W mutants displayed significant alterations in wing morphology and reduced lifespan. RNA-seq analysis of transgenic and wild-type flies revealed differentially expressed genes (DEGs), as interpreted through gene ontology analysis. Our integrated computational and genetic approach identified dMPO mutations that disrupt protein stability and alter gene expression. These findings offer new insight into how single-point mutations can lead to systemic biological changes. Author SummaryProteins are the molecular machines that drive almost every process in living organisms, and even small genetic changes can disrupt their structure and function. In this study, we focused on Curly Su, a protein in fruit flies that is similar to human myeloperoxidase. We used a computational approach called saturation mutagenesis to model over 11,000 possible single mutations in the Curly Su protein and predict how they would affect its stability. We then used genome editing to introduce several destabilizing and potential disease-causing mutations into flies. These mutant flies developed abnormal wings, had shorter lifespans, and showed widespread changes in gene activity, particularly in metabolism and immune pathways. By combining computational predictions with experimental validation, our work demonstrates a powerful and generalizable strategy for linking specific genetic mutations to whole-organism outcomes. This approach not only sheds light on the biological role of Curly Su but also provides a framework that can be applied to other genes and organisms, offering insights relevant to human health and disease.