Structural and Functional Characterization of the Leishmania donovani Ufm‐ylation Pathway

Ubiquitin fold modifier 1 (Ufm1) a ubiquitin‐like protein and Ubiquitin fold modifier activating enzyme (Uba5, E1) are proteins found in eukaryotic organisms that play a crucial role in cell cycle regulation, signal transduction, and ER stress. Leishmania donovani (Ld) is a trypanosomatid parasite that has been shown to have enzymes homologous to Uba5 and Ufm1. Leishmania Ufm1 and Uba5, as well as the substrate‐targeted proteins, are associated with the mitochondria which has not been observed in other organisms. This suggests that these Leishmania proteins may have physiological roles not yet described in other organisms. Leishmania donovani causes leishmaniasis, a disease that is accompanied by sores and lesions that will appear at varying depths of the body depending on the type and increases the host's susceptibility to co‐infection with other diseases. There are currently no effective vaccines for leishmaniasis. Uba5 and Ufm1 are critical for the metamorphosis and growth of the parasite. Therefore, biochemical study of these proteins may provide insight into the molecular basis for leishmaniasis and the fundamental role of this pathway in the parasite. We first expressed and purified LdUfm1, an N‐terminally truncated form of LdUfm1, and LdUba5. LdUfm1 and LdUfm1(tr.) were successfully purified and were characterized with SEC‐MALS. We then performed gel migration shift assays to demonstrate the compatibility of the human and Leishmania systems to conjugate LdUfm1. We found that full‐length Leishmania Ufm1 but not the truncated form were viable substrates for human Uba5, suggesting a role for the N‐terminus of Ufm1 conjugation. We are currently working to purify LdUba5 further for functional and structural comparisons to the human enzyme. Additionally, we began setting crystal trays to obtain protein crystals for structure determination. We have preliminarily obtained crystals of the truncated Ufm1. Future work will optimize these crystals, perform further experiments for structural characterization and explore the cross‐reaction between the human and Leishmania enzymes. Characterization of the Leishmania donovani conjugation pathway could facilitate the development of therapeutic treatments for leishmaniasis. Support or Funding Information 4‐VA Organization, NSF REU CHE‐1757874 This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal .