E6-associated protein (E6AP) is a member of HECT domain family of E3 ubiquitin (UB) ligase that is responsible for many biological processes. E6AP may be paired with the papillomavirus oncoprotein E6 when E6 is present in the host to recruit substrates for degradation. This may hinder the host’s antiviral response and lead to cancer development. A damaged HECT domain of E6AP has been reported in patients with Angelman Syndrome. When Legionella pneumophila enters the human body, 330 bacterial effector proteins are arranged to employ the Dot/Icm type IV secretion system, T4SS. Bacterial effector proteins: GobX, LubX, and RavN are reported to mimic the U-box E3 ligase to hijack and thrive for proliferation. Our lab has developed an orthogonal ubiquitin transfer (OUT) cascade to monitor the movement of engineered UB through the engineered E1-E2-E3 cascade. In this study, OUT cascade was utilized to profile substrates that may lead to new discoveries.