Investigating the nuclease activity of MIF and MIF-2 and their effects on DNA damage

Bulut Impraim, Buket (2024): Investigating the nuclease activity of MIF and MIF-2 and their effects on DNA damage. Dissertation, LMU München: Medizinische Fakultät

Abstract

The multifunctional inflammatory protein macrophage migration inhibitory factor (MIF) and its paralog D-dopachrome tautomerase (D-DT)/MIF-2 have important functions in various neurodegenerative diseases. This work aimed to study the relationship between the topological location of MIF and MIF-2 in different neuronal cell compartments and their functions. The focus of this study was to describe the complex modes of action of MIF and MIF-2 in the brain, as well as the discovery and function of the nuclease activity of MIF-2. In 2016, MIF was identified as a new member of the PD-D/E(X)K class of nucleases. Similar catalytic properties were discovered in this dissertation for MIF-2, where the PD-D/E(X)K nuclease domains are only partially conserved when compared with MIF. Glutamic acid at position 22 of human and mouse MIF is relevant for nuclease function. Similarly in this work, glutamic acid was identified at position 88 as being critical for the nuclease activity of human MIF-2. Moreover, the subcellular localization of MIF was found to be similar to that of MIF-2, with both homologs present in the cytoplasm, nucleus, and extracellular space, indicating a potential "moonlighting" function for MIF-2. Moonlighting proteins serve multiple functions despite originating from a single gene. Changes at catalytic sites or interactions with other proteins can cause different functions. The MIF family has different functions, both intracellularly and extracellularly. This work's second focus was the interaction between MIF and the protein apoptosis-inducing factor (AIF), since MIF is the essential nuclease in AIF-induced programmed cell death (parthanatos). AIF is assumed to escort MIF from the cytosol to the nucleus. My work detected MIF in both wild-type cells and Mif-2-deficient primary astrocyte cultures. Conversely, Mif-2 was localized to the nucleus in both wild-type cells and Mif-deficient cells. In addition, it is assumed that the translocation of MIF and MIF-2 takes place independently of one another. It has been identified that the nuclease function of MIF is an Mg2+- and Ca2+-dependent 3' exonuclease activity. In my study, MIF-2 nuclease function was inhibited using various methods, including ethylenediaminetetraacetate (EDTA), which forms chelate complexes with positively charged metal ions. The results highlight the pleiotropic effects of MIF and MIF-2, but my study focused on their roles in cellular stress and apoptosis. PARP1 inhibition has been identified as an attractive target for acute neurological diseases. Using DNA double-strand break markers and comet assays, a reduction of DNA damage was observed in Mif- and Mif-2-deficient cells compared to wild-type cells. Work in my thesis found that primary mixed neuronal cultures obtained from Mif KO animals and primary astrocytes isolated from Mif-2 KO animals showed fewer γH2AX and 53BP1 markers compared to the wild-type. This highlights the potential role of MIF and MIF-2 in DNA damage and neurodegenerative diseases. Laboratory mice and rats have long been indispensable tools in research, with mice being the by far predominant model for most diseases. This dissertation explored the unique advantages of a rat model, particularly in the context of studying the macrophage migration inhibitory factor (MIF) family. Rats, with their larger size and weight, offer significant benefits for various research applications, including investigations into MIF-related conditions like stroke and Alzheimer's disease. In this study, reverse genetic tools, including genome editing methods, were employed to establish a Mif KO rat model for investigating the critical role of MIF proteins in important biological processes. The rat model's establishment remains crucial for replicating essential insights from mouse models. In conclusion, this dissertation explored the roles of MIF and MIF-2 in neurodegenerative diseases, revealing their nuclease activity, subcellular localization, and involvement in programmed cell death together with AIF. Overall, this research enhanced our understanding of MIF and MIF-2 in neurodegenerative diseases and their potential in treating acute neurological conditions.

• Dokumententyp: Dissertationen (Dissertation, LMU München)
• Themengebiete: 600 Technik, Medizin, angewandte Wissenschaften ; 600 Technik, Medizin, angewandte Wissenschaften > 610 Medizin und Gesundheit
• Fakultäten: Medizinische Fakultät
• Sprache der Hochschulschrift: Englisch
• Datum der mündlichen Prüfung: 19. Juni 2024
• 1. Bericht­erstatter:in: Bernhagen, Jürgen