Characterization of a transgenic pig model carrying green fluorescent proteasomes

Abstract

The Ubiquitin Proteasome System (UPS) participates in many biological processes involving substrate-specific proteolysis, and can be linked to various pathologies and genetic diseases. Unfortunately, little is known about the interactions of proteasomal subunits with other sperm proteins or structures. Through a joint effort between Dr. Prather, Dr. Wells, and Dr. Sutovsky's labs, a novel transgenic pig model with Green Fluorescent Protein (GFP) fused to the 20S proteasomal core subunit α-type 1 (PSMA1) was created, hypothesizing that the PSMA1-GFP fusion protein will be incorporated into functional sperm proteasomes. The first objective of this thesis was to confirm the presence of these PSMA1-GFP fusion proteins in the transgenic boar sperm and tissues. The second objective was to identify proteasome-interacting proteins that may regulate sperm proteasomal activity during fertilization or may be the substrates of proteasomal proteolysis during fertilization. A novel interaction with seminal plasma/sperm membrane glycoprotein, MFGE8, and the 26S proteasome was revealed. Therefore, a possible role for this interaction was investigated: that the 26S proteasome mediates the degradation of MFGE8, which allows the gradual release of spermatozoa from the sperm oviductal reservoir prior to fertilization. Furthermore, GFP affinity purification was used to isolate enzymatically active proteasomes which could be used to study sperm-oocyte interactions or wherever the UPS is involved. It was concluded that this transgenic pig model can be used to study proteasome localization, proteasome interacting proteins, and tissue specific proteasome isolation in all areas of UPS research.