Disrupting the LC3 Interaction Region (LIR) Binding of Selective Autophagy Receptors Sensitizes AML Cell Lines to Cytarabine.

Suchithra Guntur,Andrew Vorobyov,Olesya Vakhrusheva,Mateusz Putyrski,Ivan Dikic,Florian Bonn,Andreas Ernst,Christian Brandts

doi:10.3389/fcell.2020.00208

Abstract

Short linear motifs (SLiMs) located in disordered regions of multidomain proteins are important for the organization of protein–protein interaction networks. By dynamic association with their binding partners, SLiMs enable assembly of multiprotein complexes, pivotal for the regulation of various aspects of cell biology in higher organisms. Despite their importance, there is a paucity of molecular tools to study SLiMs of endogenous proteins in live cells. LC3 interacting regions (LIRs), being quintessential for orchestrating diverse stages of autophagy, are a prominent example of SLiMs and mediate binding to the ubiquitin-like LC3/GABARAP family of proteins. The role of LIRs ranges from the posttranslational processing of their binding partners at early stages of autophagy to the binding of selective autophagy receptors (SARs) to the autophagosome. In order to generate tools to study LIRs in cells, we engineered high affinity binders of LIR motifs of three archetypical SARs: OPTN, p62, and NDP52. In an array of in vitro and cellular assays, the engineered binders were shown to have greatly improved affinity and specificity when compared with the endogenous LC3/GABARAP family of proteins, thus providing a unique possibility for modulating LIR interactions in living systems. We exploited these novel tools to study the impact of LIR inhibition on the fitness and the responsiveness to cytarabine treatment of THP-1 cells – a model for studying acute myeloid leukemia (AML). Our results demonstrate that inhibition of LIR of a single autophagy receptor is insufficient to sensitize the cells to cytarabine, while simultaneous inhibition of three LIR motifs in three distinct SARs reduces the IC50 of the chemotherapeutic.

Highlights

Short linear interactions motifs (SLiMs) play a crucial role in the organization and assembly of intracellular signaling complexes and have been closely associated with the development of higher order organisms (Pawson and Nash, 2003; Beltrao and Serrano, 2007)
These residues are distributed over two distinct regions localized on α1 helix, which interacts with the acidic part flanking the LC3 interacting regions (LIRs) peptides, and on β2 – α2, which is in contact with the hydrophobic part of the LIR (Figure 1B)
In region 2, we followed a similar strategy with two exceptions: in LC3B, we randomized the bulky Ile68 to allow for structural flexibility in the positioning of the α2 helix, which is in direct contact with the LIR motif

Summary

Introduction

Short linear interactions motifs (SLiMs) play a crucial role in the organization and assembly of intracellular signaling complexes and have been closely associated with the development of higher order organisms (Pawson and Nash, 2003; Beltrao and Serrano, 2007). Studies on the mutational flexibility of SLiMs and their binding partners demonstrated that interactions with altered selectivity required for the evolution of novel protein–protein networks can be established (Ernst et al, 2009; Teyra et al, 2019) To their evolutionary properties, the transient nature of SLiM interactions due to fast association/dissociation rates to their cognate binding protein is another characteristic of such interaction motifs (Sugase et al, 2007; Perkins et al, 2010). This transiency allows for an additional level of regulation, for example, by relying on multimerization of binding partners, i.e., binding avidity, or by posttranslational modification of binding site residues. For the majority of conserved SLiMs it is unclear whether a disruption of their interactions can be exploited for a therapeutic benefit

Methods

Results

Conclusion