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Using Proximity-based Proteomics to Identify Interacting Partners and Substrates for the Listerin Ubiquitin Ligase

  • Author(s): Zuzow, Nathaniel David
  • Advisor(s): Bennett, Eric J
  • et al.
Abstract

The ribosomal quality control complex (RQC) is a multiprotein complex which senses, modifies, and extracts defective protein products at the 60S ribosomal subunit for ubiquitin-mediated degradation. Central to the function of the RQC is the RING finger E3 ubiquitin ligase Ltn1, which catalyzes the ubiquitylation and subsequent degradation of defective translation products. Listerin, the mammalian ortholog of Ltn1, is crucial for early development and neuronal function, most likely preventing the formation of toxic protein aggregates in neurons.

Little is known about how Listerin functions in mammalian cells. In Chapter 1, we show that Listerin and other mammalian RQC (mRQC) components interact with each other, as well as with the ribosome. Contrary to our expectations, mRQC interactions are not induced by widespread translational stalling. We also show that translational stress causes mRQC proteins to shift into biochemical fractions known to contain the 60S ribosome.

Because detection of endogenous E3 ubiquitin ligase substrates presents a formidable challenge, traditional affinity purification and mass spectrometry (AP-MS) approaches are of limited use for this purpose. Chapter 2 details our use of tandem mass spectrometry to analyze potential Listerin substrates captured via three separate protein interaction mapping platforms: AP-MS, BioID, and APEX. Here, we describe two new classes of Listerin substrates: signaling kinases of the RSK family, and members of the tubulin glutamylation complex (TGC).

In Chapter 3, we study the functional interactions between Listerin and the putative substrates identified in Chapter 2. We show that RSK1, RSK2 and a subset of TGC proteins are ubiquitylated by Listerin in a RING-dependent manner, and that this modification is consistent with regulatory polyubiquitylation. Additionally, we observe that disruption of Listerin positively regulates mTOR via RSK-mediated inhibitory phosphorylation of TSC2, and this novel function of Listerin is possibly independent of the mRQC.

Taken together, our work demonstrates that mRQC proteins interact with each other and the ribosome, and these interactions are altered by translational stress. Our application of orthologous proteomic approaches led to the discovery of diverse Listerin substrates, opening the door to more detailed studies of this ligase with respect to human disease.

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