Characterizing new players involved in iron homeostasis during Drosophila larval development: Shifting the classic paradigm of iron metabolism

• Author / Creator

  Huynh, Nhan

• Iron plays a critical role in many biological processes. The ability of iron to gain and lose electrons allows it to be involved in a wide variety of biochemical reactions. Organismal iron is commonly found in two types of protein cofactors: heme and iron-sulfur clusters (ISCs). Iron is critical for many cellular processes, including oxygen transport, energy production, steroid hormone synthesis. Iron’s capacity for electron transfer is also a double-edged sword that can generate cell-damaging radicals. Iron-homeostasis has been linked to many diseases including hemochromatosis, the porphyrias, Friedreich ataxia, and sideroblastic anemia. Thus, iron levels have to be tightly regulated at the systemic as well as cellular levels. Many aspects of cellular iron biology remain unexplored, and many genes functions in iron metabolism still stay hidden.
  This thesis focuses on increasing understanding the regulatory mechanism by which iron and heme metabolism is coupled with steroid hormone production. I first investigated the role of AGBE in iron regulation. Prothoracic gland (PG)-knock down of this gene results in a porphyria phenotype that can be rescued in an iron-supplemented medium. Further investigation allows us to establish the relationship between AGBE, Cisd2, and IRP1A in Drosophila iron metabolism. In Drosophila, Cisd2 is required to maintain the intact ISC in IRP1A, and AGBE will act as a bridge to strengthen the interaction between Cisd2 and oxidatively damage IRP1A for the repair process.
  I also report the nuclear localization of IRP1A. This hitherto undocumented localization is tissues-specific and iron-sensitive. The unexpected presence in the nucleus suggests a function of IRP1A in gene regulation. Further work allowed me to propose a model where the nuclear IRP1A might participate in expression regulation of iron-related genes by regulating nuclear citrate level, a substrate of nuclear acetyl-CoA synthesis for histone acetylation. This finding has added an entirely unexpected aspect not explained before of holo-IRP1A in iron metabolism.
  
  I also characterized functions for a gene called ppk20 in iron or heme metabolism. This gene was identified from earlier genome-wide screening. ppk20 is a member of Drosophila epithelial sodium channel (ENaC). PG-knock down of ppk20 results in porphyria phenotype and trachea necrosis, both can result from abnormal iron homeostasis. These phenotypes can be rescued by hemin, injected ferritin, and human transferrin receptor. Thus, these data suggest a role for ppk20 in iron metabolism.
  I generated two CRISPR/Cas9 toolkits, which allow spatial and temporal gene manipulation. With these, one can generate somatic mutations, interfere with transcription or induce gene expression in the tissue of interest and at the desired time points. I also evaluated the efficiency and potential applications of another CRISPR system, Cas13, in Drosophila. Unlike Cas9, which is used to target DNA, Cas13 target RNA with high efficiency, and current preliminary data suggests its great potential in RNA targeting.

• Subjects / Keywords

  • iron
  • porphyria
  • Cas13
  • CRISPR
  • Cas9
  • mitoNEET
  • IRP1
  • fruit fly
  • glycogen

• Graduation date

  Fall 2020

• Type of Item

  Thesis

• Degree

  Doctor of Philosophy

• DOI

  https://doi.org/10.7939/r3-j6z1-7m05

• License

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