UC Riverside

The human malaria parasite remains a major public health burden in developing nations. Despite many years of research, the mechanisms controlling gene expression in the parasite are still poorly understood. While the P. falciparum genome lacks more than fifty percent of the transcription factors anticipated to regulate its 6372 genes, it encodes a large amount of genes involved in RNA metabolism and chromatin remodeling. Furthermore, preliminary data in the laboratory showed extensive nucleosome remodeling during the parasite's asexual cycle. Therefore, we hypothesized that change in chromatin structure plays an important role in controlling parasite development. To understand the role of histone post-translational modifications (PTMs) in transcriptional regulation and histone turnover, we used a shotgun proteomic approach. A total of 246 histone PTMs were identified with 126 being novel. Parasite histones were highly acetylated and methylation marks associated with transcriptional silencing were detected at low levels. To elucidate the mechanism regulating histone turnover, we treated parasite cultures with inhibitors of two distinct pathways that degrade bulk amounts of protein; the ubiquitin-proteasome system and the autophagosome-lysosome pathway. Parasites treated with inhibitors of the autophagy pathway displayed an accumulation of histone protein. The autophagy pathway was overlooked in the parasite; thus, we investigated it at the comparative genomic, cellular, biological and genetic levels. PfATG8, an autophagosome membrane marker, was detected throughout the erythrocytic stages in the apicoplast and the cytoplasm. Proteins associated with PfAtg8 were isolated by immunoprecipitation and identified by mass spectrometry. Gene ontology enrichment showed an enrichment of proteins involved with the digestive food vacuole, the phagolysosome, and the nucleus. In summary, we determined that the autophagy pathway is multifunctional and is likely involved in vesicle traffic, apicoplast biogenesis, and protein catabolism. To further validate its role in histone turnover, we took a cellular approach and colocalized histones and PfATG8 vesicles. Collectively, our work provides key information of mechanism regulating epigenetic and its effects on gene expression in the human malaria parasite.