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New insights into the N-end Rule Pathway: from the substrate recognition to its selective inhibition

  • Author / Creator
    Couto Assis Leitao, Luana
  • The N-end rule represents a crucial proteolytic pathway which regulates in vivo half-life of a protein by the identity of its N-terminal residues. As such, the instability of the substrate is conferred by the ability of the cellular machinery to recognize the N-terminal amino acid and target the protein for cellular degradation. In mammals, the recognition of the destabilizing N-terminal is mediated by the redundant E3 ubiquitin ligases UBR1/UBR2 which have two key recognition domains: UBR box domain and the N-domain. These recognition domains bind to type I primary residues (Arg, Lys, and His) and type II amino acids (Trp, Tyr, Ile, Phe, and Leu). In the first part of this study, we demonstrated that, in addition to the recognition of the N-termini by the E3 ubiquitin ligase, an N-end Rule substrate, the PKCδ has an extended half-life by the phosphorylation of Ser331, the second position residue of the cleaved fragment. Using the ubiquitin fusion method, it was shown that the PKCδ fragment was stable despite the unstable N-terminal, Asn. The findings appear to be related to the hydrophilic aspect of the phosphorylated Ser331 that disrupts the substrate binding to the NTAN1, inhibiting the ubiquitination of the fragment and subsequent protein degradation. The data presented in this study reveals that the mutation of the second position residue into an amino acid with no hydrophilic characteristics, enable the binding affinity to be reestablished. In the second part, the study is directed towards the identification of a novel inhibitor of the N-end rule pathway. Substrates of the N-end Rule Pathway have been related to several pathogeneses, such as malignant cell growth, but only a few nonselective inhibitory compounds were discovered. We have identified through half-life assays, using different reporter proteins, a novel chemical inhibitor that stabilizes type I N-terminal amino acid proteins. The selective inhibition of this proteolytic pathway could be used to suppress the degradation of specific substrates, such as pro-apoptotic protein fragments (e.g., Asp-BCL(XL), Arg-BID, and Arg-BIM(EL)). This research is the first in the field to demonstrate the selective inhibition of the Arg/N-end Rule Pathway. The PPCP and Analogs (I and II), demonstrated high specificity in the inhibition of type I N-termini with an EC50 of 3.8 µM, 1.1 µM, and 0.8 µM, respectively. Additionally, these small molecules demonstrated low cellular toxicity when used alone, appearing as great candidates for drug development. The discovery of these novel N-terminal inhibitors expands the understanding of the degradation pathway and could have a valuable therapeutic implication towards distinct malignancies.

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  • Graduation date
    Fall 2019
  • Type of Item
  • Degree
    Doctor of Philosophy
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