Case studies on protein-interaction of LSD1 SWIRM-ARDED and Afmp4p/Mp1p : pro-inflammatory mediators

Abstract

Protein-ligand and protein-protein interactions play important roles in almost all cellular processes therefore it is a general belief that understanding protein-ligand/protein interaction is essential for understanding the functions of complex biological system. This dissertation focuses on two separate investigations on protein-ligand/protein interactions. The first one examined is about the interactions between various pro-inflammatory mediators and virulence factor proteins, namely Mp1p and Afmp4p secreted by the pathogenic fungi Penicillium marneffei and Aspergillus fumigatus respectively. By investigating these protein-ligand interactions, the functional roles of these two proteins can be postulated. Previous studies found that Mp1p ligand-binding domain 2 (LBD2) can trap fatty acids and it is important for the intracellular survival of the pathogens. This shed lights on the mechanism for their virulence. Recombinant Mp1pLBD2 in Penicillium marneffei and its homolog in Aspergillus fumigatus, AFMP4, were successfully overexpressed in E. coli. Both Afmp4p and Mp1pLBD2 were proved to bind a number of ligands, including pro-inflammatory arachidonic acid and sphingolipids by NMR spectroscopy. Subsequent X-ray crystallography confirmed that arachidonic acid was embedded inside the hydrophobic cavity of these proteins. From our NMR competition titration data, arachidonic acid was shown to be able to displace palmitic acid pre-bound inside Mp1pLBD2 or Afmp4p effectively. It was also shown that the pathogenic powers of both fungi were greatly reduced after the knock-down or knock-out of the MP1 and AFMP4 genes in their corresponding fungi. In addition, the MP1 and AFMP4 genes were cloned into a non-pathogenic fungus Pichia pastoris in a knock-in experiment and this work found that the expression of either protein could prolong the intracellular survival duration of the fungi in macrophages, and thus confirming their virulence. This prolonged survival could be destroyed by the addition of arachidonic acids. Taken together all the works of our collaborators and my thesis work, it was found that the bindings of Mp1p/Afmp4p with the pro-inflammatory mediators provide a mechanism for the virulence of these proteins by harvesting the released proinflammatory precursor arachidonic acids or other sphingolipid signaling mediators from the host. Another part of my research was aimed at characterizing a protein-protein interaction between the lysine-specific demethylase 1 (LSD1) SWIRM domain and the androgen receptor DNA-binding domain (ARDBD) in the human body. The interacting surfaces on SWIRM and ARDBD were mapped and postulated, with a HADDOCK complex model provided in this thesis. The dissociation constant (KD) of this complex was determined by fluorescence spectroscopy and found to be 14.97±1.58 μM. This fluorescence study not only provides the KD, but also maps the interaction site on SWIRM. It was also known that there is a H3 N-terminus binding site on LSD1 SWIRM domain. Together with the function of providing structural stability to the LSD1 protein, the SWIRM domain is considered as a multi-functional domain vital to the catalytic function of LSD1.